Treatment of retroviral reservoirs exploiting oxidative stress

ABSTRACT

Activation of HIV-1 replication causes oxidative stress, which in turn potentiates HIV-1 replication. The common basis for the compounds of the present invention is: A) the capacity of reactivating HIV-1 from latency, and B) the ability to counteract the cellular machinery which activates in order to limit the effects of oxidative stress. In this way, oxidative stress can be potentiated and a “chain reaction” is sparked. This “chain reaction” induces a more efficient reactivation of HIV-1 from latency and, in some cases, induces selective killing of the infected cells. Actions A) and B) can either be carried out by one drug exerting both effects, or obtained by the combined use of distinct drugs. There are two main cellular machineries counteracting oxidative stress, i.e. the thioredoxin (Trx) thioredoxin reductase (TrxR) system and glutathione. Herein, we present drug strategies capable of exerting action B) by blocking either of the two machineries.

The present application relates to the use of certain compounds orcombinations of compounds in the treatment of latent viral reservoirs,particularly HIV-1. Treatment of the viral reservoirs is by theexploitation of oxidative stress: A drug repositioning approach. Thecompounds include gold-containing compounds, such as auranofin,arsenic-containing compounds, such as arsenic trioxide, and HDACinhibitors in combination with BSO.

INTRODUCTION TO THE INVENTION

Eradication of HIV-1 infection from the body has encountered exceptionaldifficulties in the presence of the latent viral reservoir, representedmainly by memory CD4⁺T-lymphocytes, which can neither be targeted bycurrent antiretroviral therapies (ART) nor recognised by the immunesystem. For this purpose, so-called “shock and kill” strategies havebeen proposed [Hamer, 2004], based on a) stimulation of viral antigenexpression in latently infected cells (i.e., the “shock” phase) in thepresence of intensified ART to suppress viral spread due to virusexpression, and b) killing of latently infected cells by the immunesystem or other means (i.e., the “kill” phase). For each of thesephases, effective drugs are being extensively searched for.

Classical drug discovery involves target discovery and validation, leadidentification by high-throughput screening, and lead optimization bymedicinal chemistry. An alternative drug development strategy is theexploitation of established drugs that have already been approved fortreatment of non-infectious diseases and whose targets are particularlyinteresting for the disease whose cure is being searched for[Dueñas-González et al., 2008]. This strategy is also denominated drugrepositioning, or indication switch [Dueñas-González et al., 2008]. Onesuccessful example of drug repositioning is furnished by chloroquine. Inaddition to its use in the antimalarial arsenal, chloroquine has beenutilized for treatment of autoimmune diseases such as rheumatoidarthritis, and is now in clinical trials as a potential agent fortreatment of certain types of cancer and viral infections [Savarino etal., 2006; Savarino et al., 2007].

For the “shock” phase of HIV-1 eradication strategies, histonedeacetylase inhibitors (HDACIs), currently in clinical trials asanticancer agents, have been proposed [Demonté et al., 2004; Mai et al.,2009]. Unfortunately, the effects of currently available compounds onHIV-1 activation from quiescence are associated with toxicity [Duvergeret al., 2009]. New HDACIs), which are epigenetic drugs inducing HIV-1escape from latency, which be class and isoform specific, have recentlybeen discovered [Mai et al., 2009]. These compounds specifically inhibitthose histone deacetylases (HDACs) belonging to the class I grouping,which are specifically involved in maintaining HIV-1 latency. However,toxicity remains a major concern. Moreover, similarly to thenon-class-specific HDACis of former generation, these drugs are not ableto induce HIV-1 activation in all of the cells within a latentlyinfected cell population. This suggests that there are differentchromatin environments maintaining HIV-1 latency in different cells.Therefore, diverse stimuli will be required to efficiently purge HIV-1from reservoirs.

In this regard, oxidative stress is thought to be an important area forvirus/host interaction. Metal ions, and other thiol-reactive chemicalspecies, may play an important role in the genesis of oxidative stress.Iron, a metal ion shown to generate hydroxyl radicals through the Fentonreaction, was found to be increased in productively HIV-1-infected cellsand to promote HIV-1 replication in vitro [Savarino et al., 1999]. Thismetal, however, is at present unlikely to find a place in HIV-1reactivating strategies, due to the side effects and the complexities ofits administration in vivo. Given the poor adsorption andbiodistribution of iron carriers, this metal is unlikely to reachsufficient plasma levels for efficient HIV-1 activation from the latentreservoirs.

Metals other than iron may cause oxidative stress. One such metal isgold [Sannella et al., 2009]. Gold-containing compounds have been shownto be useful in the treatment of rheumatoid arthritis [Patai, 1999] andhave been considered in anticancer strategies. Their proposed usefulnessin anticancer treatments is due to their antiproliferative properties.The lead structure for antitumor-active gold complexes is auranofin(FIG. 1), which is characterized by its gold(I) central atom as well asa triethylphosphine and a carbohydrate ligand. However, auranofin hasbeen shown to silence HIV activation, i.e. maintain latency (Jeon etal., 2000; Traber et al., 1999).

SUMMARY OF THE INVENTION

It is well known that activation of HIV-1 replication causes oxidativestress, which in turn potentiates HIV-1 replication. The common basisfor the compounds of the present invention is: A) the capacity ofreactivating HIV-1 from latency, and B) the ability to counteract thecellular machinery which activates in order to limit the effects ofoxidative stress. In this way, oxidative stress can be potentiated and asort of “chain reaction” is sparked. This “chain reaction” induces amore efficient reactivation of HIV-1 from latency and, in some cases,induces selective killing of the infected cells. Actions A) and B) caneither be carried out by one drug exerting both effects, or obtained bythe combined use of distinct drugs. There are two main cellularmachineries counteracting oxidative stress, i.e. the thioredoxin (Trx)thioredoxin reductase (TrxR) system and glutathione. Herein, we presentdrug strategies capable of exerting action B) by blocking either of thetwo machineries.

We have conducted experiments to test whether, and how, auranofin mightactivate HIV-1 in cell line models for HIV-1 quiescence. Surprisingly,we have shown that the gold(I)-containing compound auranofin is a potentinducer of HIV-1 activation from quiescence and is thus useful in HIV-1eradication. Auranofin shows remarkable activity in activatingreservoirs of latent retrovirus. In combination with knownantiretroviral therapy or treatment (ART), this may then be used intherapy to reduce or eliminate retrovirus infection. The effects ofauranofin on HIV-1 reactivation are particularly surprising because thisdrug was believed to act in the opposite way (silence HIV), as discussedelsewhere.

Specifically, auranofin has been found to stimulate the reproduction ofretroviruses from latent reservoirs (of said retroviruses), and this maybe used to reduce or eliminate these reservoirs in combination withconventional antiretroviral therapy. Further, histone deacetylaseinhibitors (HDACi's), iron nitriloacetate and buthionine sulfoximine haseach been found to substantially potentiate the capacity of auranofin tocombat HIV-1 latency.

We have also surprisingly found that arsenic-containing compounds andcombinations of a histone deacetylase inhibitor with a glutathionesynthesis inhibitor such as buthionine sulfoximine (BSO) are alsoeffective in treating latently infected cells. In particular, theactives are able to target and selectively kill infected, but notun-infected, cells.

Therefore, certain oxidative stress inducers are useful in the treatmentof a retroviral reservoir.

Thus, in a first aspect, there is provided the use of an oxidativestress inducer in the treatment of a retroviral reservoir, wherein saidoxidative stress inducer is a non-iron metallodrug, which is anepigeneitic modulator.

The inducer is a non-iron metallodrug epigeneitic modulator, forinstance gold-containing compounds, such as auranofin, orarsenic-containing compounds, such as arsenic trioxide, or combinationsof a histone deacetylase inhibitor with a glutathione synthesisinhibitor such as buthionine sulfoximine. Cisplatin, for instance, is amettallodrug but does not have epigenetic properties. The oxidativestress inducer may be a pro-oxidant molecule.

The present metallodrug is a compound comprising a metal ion and havingbiological activity. These metallodrugs includes metals able to induce are-arrangement of gene expression profiles within a cell. This may beexploited to induce HIV-1 activation from latency. The presentmetallodrugs may also have particular chemical properties. In general,it is preferred that they are able to release an ion carrying onepositive charge. Optionally, they may also meet particular stearicrequirements.

For example, gold(I)-containing compounds fully meet these criteria.These compounds may consist of an organic carrier and a gold(I) ion,which is released. Although this invention is not linked to anyparticular mechanisms, the atomic size of gold (approx. 174 pm) allowsinsertion in the active site of TrxR to form a complex withcysteins/selenocysteins fundamental for the biological activity of thisprotein. A three-dimensional structure can be seen in the Protein DataBank (accession number: 3H4K). In this manner, the activity of TrxR isinhibited.

The active ion of the present metallodrugs may also be a non-metal ioncapable of mimicking the gold(I) ion. In this context, somemetalloid-containing drugs, such as arsenic trioxide (As₂O₃) may beconsidered as epigenetic metallodrugs. Arsenic trioxide releases anarsenic monoxide ion carrying one positive charge and meets thestructural requirements to form covalent adducts with the cysteins orselenocystein present in reductases (atomic size of arsenic: 115 pm;atomic size of oxygen: 60 pm). A structure of arsenic oxide in complexwith a Trx suoperfamily member can be seen in the Protein Data Bank(accession: 1J9B). This structure strongly suggests that one similaradduct is formed with thioredoxin reductase.

The ion released by the present metallodrug may thus not be derived fromplatinum, the oxidation of which can only result in Pt(II) or Opt(IV)ions, or iron, from which Fe(II) or Fe(III) ions are derived and has ashorter atomic size as compared to gold. Thus, metallodrugs comprisingiron ions or platinum ions are excluded from the present invention andare considered only if combined with one of the aforementionedmetallodrugs of the invention. Particularly preferred examples arecompounds comprising gold, preferably gold (I), or arsenic ions.Preferred gold-containing compounds include gold salts or goldderivatives. Auranofin is particularly preferred. Preferredarsenic-containing compounds include arsenicals such as arsenic oxides,which include white arsenic (As₂O₃, but may also be found as As₄O₆).Arsenic trioxide (As₂O₃) is particularly preferred. Another commonactivity shared by gold(I)-containing compounds and arsenic trioxide isthe capacity to act as superoxide dismutase mimics, thus facilitatingintracellular production of radical oxygen species (ROS). ROS arelargely known to activate HIV-1 from latency.

Epigeneitic modulators are known in the art to play a role in DNAmethylation and chromatin remodelling, i.e. in DNA winding and/orunwinding, thereby modulating gene expression.

The non-iron metallodrug epigeneitic modulator is capable of inducingoxidative stress. It is also preferably capable of inhibitingthioredoxin reductase (TrxR) and/or acting as a superoxide dismutase(SOD) mimic. The metallodrug preferably inhibits thioredoxin reductaseby to blocking its active site. This may be achieved by complexingdirectly with the selenocystein residue known to be important for thereducing activity of these proteins. The metallodrug may also suppresssynthesis of TrxRs.

Pro-oxidant molecules are also envisaged, particularly inhibitors ofgamma-glutamyl cysteine synthetase, a limiting enzyme in the glutathionesynthetic pathway [Anderson, 1998]. Preferred inhibitors of this enzymeinclude buthionine sulfoximine (BSO), an irreversible inhibitor. BSO isthus a glutathione synthesis inhibitor and such inhibitors are alsopreferred. The inhibitor is most preferably provided in combination witha histone deacetylase inhibitor (HDACi).

The compounds of the present invention (which can include combinationssuch as a glutathione synthesis inhibitor with an HDACi) are capable ofselective killing. This is the ability to target and destroy latentlyinfected cells, but not uninfected cells. In other words, cellscomprising viral reservoirs are targeted but uninfected cells are notdestroyed, leading to an advantageous reduction in side effects. It ispreferred that the target cells are transitional memory T-CD4⁺ cells(T_(TM)s).or the central memory T CD4⁺ cells (T_(CM)s) These are theprincipal reservoir for HIV-1 latency in individuals underantiretroviral therapy (ART) and presenting low CD4 counts [Chomont etal., 2009]. the central memory T CD4⁺ cells (Tcms) are precursors ofT_(TM)s and represent a more stable HIV-1 reservoir. Thus, the presentinvention is particularly useful in treating patients with viralreservoirs, especially those patients who are presenting low CD4 counts.Said patients should be undergoing, or have undergone, antiretroviraltherapy (ART) as this can assist in preventing the newly formed virusinfecting other cells.

Also provided is a method of treating a patient suspected of having aretroviral reservoir, comprising administering to said patient anoxidative stress inducer as defined herein, which may include a non-ironmetallodrug epigenetic modulator or an HDACi together with a glutathionesynthesis inhibitor, such as buthionine sulfoximine. The method mayfurther comprise administering at least one of a (further) histonedeacetylase inhibitor (HDACi), BSO, gold-containing compound, such asauranofin, an arsenic-containing compound, such as arsenic trioxide(As₂O₃) and/or iron nitriloacetate or ferrous sulphate.

The invention also provides a method of selectively targeting cellslatently infected by a retrovirus, said method comprising contacting thecells with said oxidative stress inducer.

Thus, we provide strategies capable of not only activating HIV-1 fromlatency, but also of counteracting the cellular antioxidant machinerymaintaining HIV-1 in a quiescent state. In case of epigeneticmetallodrugs, both activities are carried out by the same drug(induction of ROS activating HIV-1 replication and inhibition of thecellular antioxidant protein TrxR). In case of the HDACI plus BSOcombination, these effects are exerted by separate drugs (an HDACiinducing epigenetic HIV-1 reactivation, and BSO inhibiting the synthesisof the cellular reducing peptide, glutathione).

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1. The structure of auranofin [gold(1+);3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate;triethylphosphanium].

FIG. 2. Dose-dependent stimulation of HIV-1 replication by auranofin inACH-2 cells.

The graph shows the concentration-dependent stimulation of HIV-1 p24production in ACH-2 cells at Day 3 of incubation with the drugs. x axis:Drug concentration; y axis: fold-increase in HIV-1 p24 (Log transform ofthe percentage of baseline levels in untreated cultures). The line orcurve best fitting the data points is shown.

FIG. 3. Combined effects of auranofin (0.25 □M) and MC2113 (1□M) onHIV-1 replication in U1 cells. U1 cells were incubated with either drugalone, or in combination, and p24 production was assessed at 24 h oftreatment. In this case, the synergistic effect was so evident that itdid not need analysis using percentage-of-synergism surfaces.

FIG. 4. Dose-dependent induction of LTR-controlled expression ofgreen-fluorescent protein (GFP) by auranofin. We used quiescentlyinfected T-lymphoid Jurkat cell clone, established by Jordan et al.(2003). This clone, namely 8.4, contains the entire HIV-1 genome undercontrol of the LTR and present the GFP gene replacing nef. The 8.4 cellsdisplay non-significant basal levels of GFP expression. Cells wereincubated with the different treatments, and GFP expression wasmonitored in gated live cells at 72 h by standard flow cytometrictechniques. Results are presented as fluorescence histograms. Eachhistogram reports the percentage of fluorescent cells beyond a thresholdvalue established using non-infected Jurkat cells.

FIG. 5. Induction of LTR-controlled expression of green-fluorescentprotein (GFP) by auranofin and a class I histone deacetylase inhibitor(MC2113). Quiescently infected Jurkat 8.4 cells were treated with aclinically relevant concentration of auranofin (0.25 microM) or 1 microMof MC2113, or both. Data are presented as in FIG. 4.

FIG. 6. Induction of NF-kappaB (p65/p50) nuclear transportation byauranofin at different incubation times. Quiescently infected Jurkat 8.4cells were incubated with auranofin a clinically relevant concentrationof auranofin (0.25 microM), and the presence of p65 in nuclear extractswas quantitated by a nuclear factor colorimetric assay. Results arepresented as a percentage of the signal obtained in nuclear extractsfrom cells incubated with TNF-alpha for 1.5 h.

FIG. 7. Combined effects of auranofin and iron nitriloacetate (FeNTA)resulting in synergistic stimulation of HIV-1 replication in ACH-2cells. 3D surface showing synergism between drugs. x,y axes: drugconcentration; z axis: percentage of synergism between the two drugs.Percentage-of-synergism values represent the percent difference betweenthe effects of the drug combination and the sum of the effects ofauranofin and FeNTA administered separately at matched concentrations,calculated as follows:

PS=100·[E _(drug A+drug B)−(E _(drug A) +E _(drug B))]/(E_(drug A) +E_(drug B))

where PS is the percentage of synergism and E is the effect of the drugconcentration, expressed as the fold-increase in p24 production.

FIG. 8. Combined effects of auranofin and buthionine sulfoximine (BSO)resulting in synergistic stimulation of HIV-1 replication in ACH-2cells. For figure interpretation the reader is addressed to caption ofFIG. 7.

FIG. 9. Cell killing by histone deacetylase inhibitors (HDACi).

Panels A-B: Correlation between HIV-1 p 24 production in HDACi-treatedlatently infected ACH-2 (panel A) and U1 (panel B) cells and inhibitionof infected cell viability. Cells were incubated with the test compounds(1 □M), and p24 production was measured by ELISA in cell culturesupernatants. After collection of supernatants, cell viability wasmeasured by the highly standardised methyl tetrazolium (MTT) method. xaxis: fold-increase in HIV-1 p24; data are presented as a Log transformof the percentage of baseline levels in untreated cultures. y axis:percentage reduction in cell viability in comparison to untreatedcontrols incubated under similar conditions. Panels C-D: Selectivekilling of HIV-1 infected lymphocytic (panel C) and monocytic (panel D)cell cultures by MC 1855. Non-infected H9, and U937, and HIV-1 infectedH9_(IIIB), ACH-2, and U1 cells were incubated with the test compoundsfor seven days, and cell viability was measured as described above. xaxis: drug concentration. y axis: percentage reduction in cell viabilityin comparison to untreated controls.

FIG. 10. Dose-dependent stimulation of HIV-1 replication by MS 275 inACH-2 cells.

The graph shows the concentration-dependent stimulation of HIV-1 p24production in ACH-2 cells at Day 3 of incubation with the drug. x axis:Drug concentration; y axis: fold-increase in HIV-1 p24 (Log transform ofthe percentage of baseline levels in untreated cultures). Appropriatetransformations were adopted to normalise the data where necessary. Theline or curve best fitting the data points is shown.

FIGS. 11 and 12. Synergism of histone deacetylase inhibitors (HDACi)with buthionine sulfoximine (BSO).

The three dimensional (3D) graph shows the 3D surface of synergismbetween each HDACi and BSO. x,y axes: drug concentration; z axis:percentage of synergism between the two drugs. Percentage-of-synergismvalues represent the percent difference between the effects of the drugcombination and the sum of the effects of MS 275 and BSO administeredseparately at matched concentrations, calculated as follows:

PS=100·[E _(drug A+drug B) −(E _(drug A) +E _(drug B))]/(E _(drug A) +E_(drug B))

where PS is the percentage of synergism and E is the effect of the drugconcentration, expressed as the fold-increase in p24 production.

FIG. 13. Stimulation of HIV-1 LTR-controlled expression of greenfluorescent protein (GFP) by MS-275 and buthionine sulfoximine (BSO),alone or in combination in a Jurkat cell clone (A1). The A1 cell clone,derived from T-lymphoid

Jurkat cells, established by Jordan et al. as a model for latent HIV-1infection. This clone has an integrated GFP/Tat construct under controlof the HIV-1 LTR and displays a basal proportion of cells expressingGFP, which increase following stimuli activating the HIV-1 promoter. A1cells were incubated for 72 h with the different treatments, and GFPexpression was monitored by standard flow-cytometric techniques andassessed as the percentage of fluorescent cells (indicated for eachhistogram) beyond the threshold value established using controlnon-transfected Jurkat cells. One experiment out of three with similarresults. The histograms derived from double-drug treatments were foundto be significantly different (P<0.01) from those derived fromtreatments with the single drugs at matched concentrations(Kolmogorov-Smirnoff statistics).

FIG. 14. Effects of HDAC inhibitor, MS-275 and buthionine sulfoximine(BSO), alone or in combination. Cell viability values are given at 72 hof incubation, as determined by the methyl tetrazolium (MTT) method:ACH-2 cells (A), Jurkat 6.3 cells (B), uninfected Jurkat cells (C).Results are presented as percentages of the absorbance (λ=550) inuntreated controls subtracted of background (means±SEM; 3 experiments).Asterisks show the significant differences found between BSO treatmentsand matched treatments in the absence of BSO (*P<0.05; **P<0.01;***P<0.001). Statistical significance was calculated usingrepeated-measures, two-way ANOVA and Bonferroni's post-test, followingan appropriate transformation to restore normality, where necessary.

FIG. 15. Dose-dependent induction of LTR-controlled expression ofgreen-fluorescent protein (GFP) by arsenic trioxide. In this experiment,we used the T-lymphoid Jurkat cell clone A1, which has an integratedGFP/Tat construct under control of the HIV-1 LTR. Cells were incubatedwith the different treatments, and GFP expression was monitored in gatedlive cells at 72 h by standard flow cytometric techniques. Results arepresented as fluorescence histograms. Each histogram reports thepercentage of fluorescent cells beyond a threshold value establishedusing non-infected Jurkat cells.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect, there is provided the use of a gold-containing compound inthe treatment of a retroviral reservoir.

It is preferred that the gold-containing compound is capable of inducingretroviral replication in a recognised model of a reservoir of saidretrovirus. In the case of HIV-1, this model may be selected from the U1and ACH-2, for example. It is further preferred that compounds of thepresent invention have a minimum activity in the U1 assay describedhereinunder in the accompanying Experimental section of around a 500%fold increase (%).

The preferred retrovirus is a simian or human lentivirus, such as HIV,for example. HIV-1 is the preferred target of the present invention.

Preferably, the gold-containing compound is auranofin (FIG. 1), which ischaracterized by its gold(I) central atom as well as a triethylphosphineand a carbohydrate ligand. Other related and preferred gold complexesinclude, for example, the chloro analogue: Et₃PAuCl and gold thiomalate.These may have multiple modes of action which are still being explored.Auranofin was recently shown to induce a shift towards the pro-oxidantside of the intracellular redox potential [Sannella et al., 2008]. Theactive species is likely the gold ion itself and the ligands are morerelevant for the biodistribution and kinetic properties of the agents.Ideal candidates for HIV-1 eradicating strategies should not induceimmune activation (detrimental for HIV-1-infected individuals [Savarinoet al., 2000]). It is, therefore, advantageous that organic gold saltsare endowed with anti-inflammatory properties.

The gold (I) or (II) ion is, therefore, bioavailable. It will beappreciated that the gold-containing compound can also be referred to asa complex. The gold-containing compound may comprise gold in its (I) or(II) oxidation state, with (I) being particularly preferred. Thegold-containing compounds of the invention are ionic chemical compoundsof gold or organogold compounds.

Preferably, the gold-containing compound can be used either alone or incombination with at least one of an HDACi, BSO and/or ironnitoloacetate. Combinations with BSO are preferred and combinations withat least one HDACi are particularly preferred.

The invention also provides the use of an oxidative stress inducer,which is a non-iron metallodrug epigeneitic modulator, in combinationwith at least one HDACi in the treatment of a retroviral reservoir. Theoxidative stress inducer is most preferably a gold- orarsenic-containing compound, such as auranofin or arsenic trioxide, or aglutathione synthesis inhibitor, such as BSO. Iron Nitroloactate is alsoenvisaged in combination with an HDACi.

To the extent that HDACi's may be considered as oxidative stressinducers, the oxidative stress inducer mentioned above is a non-HDACioxidative stress inducer. In other words, the invention may provide theuse at least one HDAC in combination with at least one other (non-HDACi)oxidative stress inducer in the treatment of a retroviral reservoir.Said other (non-HDACi) oxidative stress inducer may be auranofin or BSO,iron nitriloacetate or ferrous sulphate, for instance.

Methods of treatment, corresponding to the present uses are alsoenvisaged.

It will be appreciated that reference herein to iron nitriloacetateapplies to range of iron-containing compounds having a similar activityin vitro or in vivo. Another suitable example is ferrous sulphate. Theterms are thus interchangeable unless otherwise apparent.

Oxidative stress was shown to be linked to HIV-1 replication by binaryinteractions, with HIV-1 replication sparking oxidative stress, andoxidative stress activating HIV-1 from quiescence [Israel andGougerot-Pocidalo, 1997]. The mechanisms behind the oxidativestress-induced HIV-1 activation from quiescence are numerous and arestill poorly explored. Oxidative stress was shown to drive the balancebetween the activities of histone deacetylases (HDACs) and histoneacetyl transferases (HATs) towards an increased HAT activity [Rahman etal., 2004]. This favors DNA unwinding and transcription of severalgenes, including the HIV-1 provirus. Nevertheless, the use of oxidativestress inducers in combination with HDACi's has not been contemplated.

Auranofin may not necessarily induce HIV-1 reactivation throughoxidative stress, it may act via an alternative mechanism. We have shownthat auranofin induces HIV-1 activation from quiescence, likely by anovel mechanism. Evidence that this drug activates HIV-1 from quiescenceis derived from its reproducible effects in four different cell lines inwhich LTR-driven gene expression is inducible. The view that auranofinactivates HIV-1 gene expression by a novel mechanism is suggested by itseffects in combination with drugs known to activate HIV-1 genetranscription by different and well characterized mechanisms, and issupported by the extant literature on its intracellular targets[Rigobello et al., 2002; Rigobello et al., 2005; Omata et al., 2006;Talbot et al., 2008].

Auranofin was shown to be an inhibitor of TrxRs, which areselenoproteins involved in maintenance of the intracellular redoxhomeostasis [Rigobello et al., 2002; Rigobello et al., 2005; Omata etal., 2006]. Auranofin was also shown to inhibit the synthesis of TrxRs[Talbot et al., 2008]. TrxRs are found in two main isoforms, onecytosolic (TrxR1) and one mitochondrial (TrxR2) [Lu and Holmgren, 2009].TrxRs have several substrates, the principal of which is thioredoxin(Trx1 is the cellular isoform; Trx2 is the mitochondrial isoform). TrxRsmaintain Trx in a reduced state which in turn reduces severalintracellular proteins. Apart from Trx, TrxRs also reduce other targetsincluding HIV-1 Tat, which is inactivated by action of TrxR (Kalantariet al., 2008).

In light of this evidence, one could surmise that the effects ofauranofin on HIV-1 reactivation could be mediated by Tat. Auranofin,however, carried out HIV-1 inducing effects in cell lines such as ACH-2and U1, which have a defective Tat/TAR axis, thus suggesting that othertargets are involved in the HIV-1 inducing effects of auranofin.Auranofin, in fact, was shown to act on multiple intracellular targets.Apart from its effects on selenoproteins, this drug was found to inhibitsome kinases such as protein kinase C [Daniel et al., 1995], cathepsins[Chircorian and Barrios, 2004].

In our study, auranofin potently enhanced the HIV-1 activating effectsof FeNTA, which generates reactive oxygen species (ROIs) through theFenton reaction. Moreover, the effects of auranofin were enhanced byBSO, a compound that induces glutathione depletion and thereforedecreases the ability of cells to counteract oxidative stress. If, asother authors (Sannella et al., 2009) report, auranofin inducespro-oxidant effects, its mechanism may be distinct from those of otherpro-oxidant molecules such as FeNTA and BSO. The HIV-1 activating effectof auranofin alone is herein supported by its NF-kappaB activatingeffects. Oxidative stress was shown to induce NF-kappaB (p65/p50)nuclear translocation [Rahman et al., 2004]. This nuclear factor bindsto specific sites on the HIV-1 LTR and promotes transcription of theproviral genome [Williams et al., 2007].

In this regard, we found in the present study that auranofin inducesNF-kappaB nuclear translocation and DNA binding under conditions similarto those at which it induces HIV-1 replication. This was a reallysurprising finding, in light of previous reports [Jeon et al., 2000;Traber et al., 1999]. Apparent discrepancies between our results andthose of previous studies showing an inhibitory effect of auranofin andother gold-containing compounds on NF-kappaB activation [Jeon et al.,2000; Traber et al., 1999], can be reconciled by considering thedifferent drug concentrations adopted. The auranofin concentrationsadopted in the previous studies to show NF-kappaB inhibition wereapprox. two orders of magnitude superior to those adopted in the presentstudy. Such drug concentrations, which are superior to those clinicallyachievable in the treatment of rheumatoid arthritis, were toxic to ourcell lines (data not shown). Instead, we have shown that auranofin canhave the opposite effect to HIV silencing, namely activation. This isparticularly the case at suitable concentrations, as will be easilydetermined by the skilled person. By way of guidance, however, it ispreferred to use a 0.125-0.5 microM range of concentrations,approximating the mean plasma levels observed during treatment ofrheumatoid arthritis [Benn et al., 1991, herein incorporated byreference]. Other preferred ranges include 0.1-0.6, 0.125-0.3, 0.2-0.6,0.2-0.7, 0.125-0.2 and 0.125-0.175 microM.

The skilled person will appreciate that these can be upscaled to humandosages, but any one of the following ranges is preferred: 0.025-0.2mg/kg/day, 0.02-0.3 mg/kg/day, 0.01-0.3 mg/kg/day, 0.005-0.3 mg/kg/day,0.03-0.2 mg/kg/day, 0.03-0.4 mg/kg/day, 0.025-0.4 mg/kg/day and 0.02-0.5mg/kg/day. A dosage range of 0.025-02 mg/kg/day is particularlypreferred.

The auranofin-induced NF-kappaB nuclear translocation, though furthersupporting the view that auranofin activates HIV-1 from quiescence byinduction of an oxidative stress, cannot be taken as an evidencepointing to NF-kappaB as the main effector for the effects of auranofinon HIV-1 replication. Several other transcriptional factors potentiallyactive on the HIV-1 LTR are activated by oxidative stress [Wu et al.,2004]. Moreover, oxidative stress switches the balance between HDAC andHAT activities towards increased activity of HATs. In this regard, wefound synergistic effect of auranofin and HDACIs.

Although gold ions (I and II) alone are poor Fenton catalysts, organiccomplexes of gold may enhance the Fenton reaction catalyzed by Fe²⁺ byacting as superoxide dismutase (SOD) mimics [Huang et al., 2005]. SODmimics, by catalyzing the superoxide/peroxide conversion, may replenishthe intracellular hydrogen peroxide pools consumed by the Fentoncatalyst, thus furnishing new substrate to the Fenton reaction. SODmimicry and TrxR inhibition that are not necessarily mutually exclusiveand may have a common background. Thus, it is possible to hypothesizethat both mechanisms cooperate to activate HIV-1 from quiescence.Finally, we cannot exclude that other, as yet unexplored mechanisms mayunderlie the HIV-1 activating effect of auranofin.

The results of the present study suggest a new application for existingdrugs in induction of HIV-1 activation from quiescence. It also suggestsnovel strategies based on two-drug combinations activating HIV-1 atnon-toxic drug concentrations. Auranofin is a drug successfully used fortreatment of rheumatoid arthritis and leukemia was found in the presentstudy to induce HIV-1 activation from quiescence at clinicallyachievable concentrations, which have a toxicity profile that iswell-characterized and shown not to endanger human health. Moreover, weshow that effective auranofin concentrations can be further decreased byconcomitant use of other HIV-1 activating agents such as HDACIs, actingby different mechanisms.

Our results showed that auranofin induced a time- and dose-dependentHIV-1 reactivation (P=0.0295, t-test for regression; FIG. 5B) in the0.125-0.5 microM range of concentrations, approximating the mean plasmalevels observed during treatment of rheumatoid arthritis [Benn et al.,1991]. In line with its capacity to induce HIV-1 activation, auranofininduced nuclear translocation of NF-kappaB, an important transcriptionfactor for HIV-1. The effects of auranofin on HIV-1 activation fromquiescence were additive or synergistic with those of other compoundsenhancing HIV-1 replication. These included histone deacetylaseinhibitors (HDACIs), which favor HIV-1 transcription by epigeneticregulation of DNA unwinding, iron nitriloacetate, which promotes HIV-1transcription by intracellular generation of reactive oxygen species(ROIs), and buthionine sulfoximine, a glutathione synthesis inhibitorthat impairs the cell's capacity to counteract oxidative stress. Thesecombined effects allow use of both auranofin and each of theaforementioned drugs at concentrations that are non-toxic for uninfectedcells.

Finding possible cures for HIV-1/AIDS, capable of eradicating the virusfrom the body, is a major scientific challenge for the 21st century. Onefurther avenue of investigation is aimed at investigating potentialdrugs and drug combinations useful for the elimination of latent HIV-1reservoirs that persist despite antiretroviral therapy (ART). Thisinvolves overcoming the latent barrier by inducing the replication ofHIV in latently infected T cells while preventing the spread of thenewly produced virions to uninfected cells by providing ARTsimultaneously. Histone deacetylase inhibitors (HDACi's) have beenpostulated to be potentially useful tools in HIV-1 eradicationstrategies [Demonté et al., 2004], and valproic acid (VA), a relativelyweak HDACi, was shown to promote HIV-1 escape from latency in vitro andto reduce the numbers of latently infected memory CD4+cells in vivo incombination with antiretroviral therapy [Lehrman et al., 2005; Smith,2005]. Such strategies have been dubbed “shock and kill” [Hamer, 2004].The low potency of VA (EC₅₀ in the millimolar range) is likely to havecontributed to its failure to induce HIV-1 eradication.

Novel and more potent HDACi have been developed for inducingdifferentiation in tumours [Mottet and Castronovo, 2008]. Many of thenew agents, however, are non-specific inhibitors for all types of HDAC,which variously play important roles in the cell cycle [Dokmanovic etal., 2007]. Class I HDACs comprise HDAC1-3 and 8, are predominantlynuclear enzymes and are ubiquitously expressed [Annemieke et al., 2003].Class II HDACs comprise HDAC4-7,9 and 10 and shuttle between the nucleusand the cytoplasm [Annemieke et al., 2003]. HDAC1 likely maintains HIV-1latency by acting in a multi-molecular complex with c-Myc and LTRs[Williams et al., 2006; Jiang et al., 2007].

Other strategies have been explored for inducing HIV-1 escape fromlatency, including use of the natural substance prostratin (whosemechanisms are, so far, largely unexplored) and diacyl glycerol lactonesthat interfere with T-cell activation [Hezareth, 2005; Hamer; 2004].

Oxidative stress is another potent means promoting HIV-1 replication[Hulgan et al., 2003; Savarino et al.; 1999; Garaci et al., 1997;Palamara et al., 1996]. Reactive oxygen intermediates promote activationand nuclear translocation of nuclear factor-kappaB (NF-kappaB) [Bowieand O'Neill, 2000], a transcription factor enhancing HIV-1 transcriptionand replication, which can be inhibited by high concentrations ofglutathione and other antioxidants [Palamara et al. 1996]. Smallmolecule redox state modulators have, so far, been poorly explored fortheir HIV-1 eradication potential.

We have now, surprisingly, found that two types of HDACi inhibitor, thebenzamides and the hydroxamates, show remarkable activity in activatingreservoirs of latent retrovirus preferably in combination with theglutathione-synthesis inhibitor, buthionine sulfoximine (BSO). Incombination with known antiretroviral therapy or treatment (ART), thismay then be used in therapy to reduce or eliminate retrovirus infection.

Specifically, benzamide and hydroxamate inhibitors of histonedeacetylases have been found to stimulate the reproduction ofretroviruses from latent reservoirs thereof, and this may be used toreduce or eliminate these reservoirs in combination with conventionalantiretroviral therapy. Further, buthionine sulfoximine (BSO) has beenfound to substantially potentiate the anti-retroviral activity of thebenzamide HDACi's. Analogues of BSO are also preferred.

In an aspect of the invention, there is provided the use of a histonedeacetylase inhibitor (HDACi) of either the benzamide or the hydroxamatevariety in the treatment of a retroviral reservoir. It is particularlypreferred that the HDACi is used in combination with BSO.

It is preferred that the inhibitor is capable of inducing retroviralreplication in a recognised model of a reservoir of said retrovirus. Inthe case of HIV-1, this model may be selected from the U1 and ACH-2, forexample. It is further preferred that compounds of the present inventionhave a minimum activity in the U1 assay described hereinunder in theaccompanying Experimental section of 800 fold increase (%).

As with gold-containing compounds, the preferred retrovirus is a simianor human lentivirus, such as HIV-1, for example, and HIV-1 is thepreferred target.

Thus, HDACi's may be used in a number of aspects of the invention, forinstance with oxidative stress inducers or. The oxidative stress inducermay include gold-containing compounds, such as auranofin, arseniccompounds, such as arsenic trioxide, or glutathione synthesisinhibitors, such as BSO. Preferred HDACi's for use in all aspects of theinvention as described below.

So-called “classical” HDIs act on Class I and Class II HistoneDeacetylases. The classical HDACi bind to the zinc containing catalyticdomain of the HDACs. These classical HDIs fall into several groupings.These include hyroxamic acids, such as Trichostatin A; cyclictetrapeptides (such as trapoxin B), and the depsipeptides; benzamides;electrophilic ketones; and aliphatic acid compounds such asphenylbutyrate and valproic acid. So-called “Second generation” HDIsinclude SAHA/Vorinostat, Belinostat/PXD101, MS275, LAQ824/LBH589, CI994,and MGCD0103. The sirtuin Class III HDACs are NAD+ dependent and aretherefore inhibited by nicotinamide, as well derivatives of NAD,dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.Suberoylanilide Hydroxamic Acid (SAHA) particularly preferred,especially in combination with gold-containing compounds such asauranofin.

Particularly preferred HDACi's are benzamide HDACi's. These may have theformula:

in which Y comprises one or two six-membered rings, each beingunsaturated or partially unsaturated and heterocyclic or homocyclic,with two to eight linking atoms, and wherein either the linking atoms ora ring comprises an amino group and a carbonyl group.

More preferred benzamides are the close analogues of the compoundsidentified as MC 2211, MC2113 and MS 275 below are particularlypreferred, and MS 2113is most preferred.

MC 2211 is:

MC2113 is:

MS 275, orN-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyhaminomethyl]-benzamide),is disclosed in EP1626719, and has the structure:

Preferred hydroxamate HDACi's have the formula:

wherein R is a direct bond or an ethylene or ethenylene group, X is ═CH—or ═N—, R¹ is aryl or heteroaryl, and is mono- or bi-cyclic, and R² isstraight or branched chain alkyl, optionally substituted by a mono- orbi-cyclic aryl or heteroaryl group. Preferably, R² is unsubstitutedalkyl, preferably methyl, ethyl, or isopropyl. R¹ is preferably phenylor naphthyl. R is preferably ethenylene. X is preferably ═CH—. PreferredHDACi's are the Class 1 HDACi's.

Also preferred are hydroxamate HDACi's such as SuberoylanilideHydroxamic Acid (SAHA). This is a hydroxamic acid-containing hybridpolar molecule and specifically binds to and inhibits the activity ofhistone deacetylase. SAHA is known in the art to exhibit an antitumoreffect by increasing expression of genes regulating tumor survival andto SAHA reduce the production of proinflammatory cytokines in vivo andin vitro (Mascagni et al, “The antitumor histone deacetylase inhibitorsuberoylanilide hydroxamic acid exhibits antiinflammatory properties viasuppression of cytokines” PNAS Mar. 5, 2002 vol. 99 no. 5 2995-3000).

The use of a histone deacetylase inhibitor (HDACi) in the treatment of aretroviral reservoir is also envisaged. The inhibitor may be selectedfrom the HDACi's discussed herein and preferably:

-   -   a) a benzamide HDAC inhibitor having the formula:

in which Y comprises one or two six-membered rings, each beingunsaturated or partially unsaturated and heterocyclic or homocyclic,with two to eight linking atoms, and wherein either the linking atoms ora ring comprises an amino group and a carbonyl group; or

-   -   b) a hydroxamate HDAC inhibitor having the formula:

wherein R is a direct bond or an ethylene or ethenylene group, X is═CH—or ═N—, R¹ is aryl or heteroaryl, and is mono- or bi-cyclic, and R²is straight or branched chain alkyl, optionally substituted by a mono-or bi-cyclic aryl or heteroaryl group. Preferably, the HDACi is a classI HDACi.

Preferably, the inhibitor is capable of inducing retroviral replicationin a recognised model of a reservoir of said retrovirus. The retrovirusmay be an HIV virus, preferably HIV-1 and the model may be selected fromthe U1 and ACH-2 cellular models.

Preferably, the benzamide is a close analogue of one of the compoundsidentified as MC 2211 and MS 275 herein. The inhibitor may be a compoundhaving the formula:

Preferably, R² is an unsubstituted alkyl, preferably methyl, ethyl, orisopropyl. Preferably, R¹ is phenyl or naphthyl. R is preferablyethenylene. X is preferably ═CH—.

It is preferred that the inhibitor is a Class 1 inhibitor and is notnon-specific. Preferably, the treatment includes, in addition,anti-retroviral therapy.

The use of a histone deacetylase inhibitor (HDACI), particularly of thebenzamide variety, in combination with a glutathione synthesisinhibitor, such as BSO (buthionine sulfoximine), in the treatment of aretroviral reservoir is also provided. Preferably, the inhibitor isN-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyhaminomethyl]-benzamide).It is preferred that the BSO and benzamide HDACi are administeredtogether.

The accompanying Example 2 provides suitable techniques to establishwhich Class of HDAC compounds of the invention inhibit. It isparticularly preferred that the HDACi's of the invention are notnon-specific HDACi's.

The HDACi's of the present invention may be used in the treatment ofpatients suspected of having latent reservoirs of retroviruses. Suchretroviruses will be referred to herein as HIV-1, although it will beappreciated that this is for convenience, and that all such referencesinclude references to other retroviruses, unless otherwise apparent fromthe context.

The treatment is preferably intended to eliminate any latent reservoirof retrovirus. However, it will be appreciated that such treatments mayalso be used, less preferably, to control latent retrovirus. This may beadvantageous where an individual is particularly susceptible to repeatinfections from the reservoirs, for example.

A latent reservoir is a cell or cells containing the retrovirus eitherin quiescence, or replicating at low rates, such as less than 5% of thenormal rate, such that the cell can act to protect the virus for aperiod of time, and often only releases viable virions weeks or monthsafter normal antiretroviral treatment has stopped, thereby requiringcontinued treatment of the individual, in case infection recurs throughsuch a reservoir.

The ‘normal’ antiretroviral treatment referred to above, and which isadministered to HIV-1-infected individuals, generally consists of acombination of at least three different drugs belonging to differentclasses, and are normally selected from the nucleosidic/nucleotidicreverse transcriptase inhibitors (NRTI's), the non-nucleosidic reversetranscriptase inhibitors (NNRTI's), and the protease inhibitors (PIs). Afusion inhibitor may also form a part of the treatment. Recently, afurther drug class, the integrase inhibitors, has been added to thearsenal of antiretroviral drugs. Antiretroviral drug combinations, ingeneral, show the capacity to block ongoing viral replication, but haveno potential for eradicating the virus from the body.

For the elimination of retroviral reservoirs, current strategiespostulate the use of HDACi's, especially the Class 1 HDACi's, togetherwith conventional anti-retroviral therapy, as well known in the art, andas described above, for example [Savarino et al. 2009].

WO2007/121429 (Gladstone Institute), WO 03/053468 (Univ Libre Bruxelles)relate to uses of HDACi's. various HDACi structures are disclosed in WO2004/069823 (Methylgene, Inc) and WO 2004/103369 (Schering AG). Munier Set al (Retrovirology, 23, Nov. 2005, 2:73) describes characterisation oftwo candidate genes, NCoA3 and IRF8, which may be involved in thecontrol of HIV-1 latency. Garaci et al (Journal of Leukocyte Biology,July 1997, Vol 62, No.1, pp 54-59) describes certain uses of BSO.

The HDACi may be administered direct to the patient in any suitable,pharmaceutically acceptable vehicle, and by any suitable route, as maybe determined by a skilled person or physician. The HDACi may beadministered as an injection or by transdermal patch, for example, andmay be in free solution, or bound to a carrier. Other formulations, suchas tablets, suppositories, creams and sprays will generally be lessuseful, although they may be used if deemed appropriate.

Suitable vehicles may include targeted liposomes, bearing suitableantibodies, but infected latent cells are largely associated with thecirculation, so administration by intravenous injection is aparticularly preferred route.

The present invention also provides the novel compounds of Table 1 and,independently, methods for making such, as illustrated in theaccompanying Experimental section.

The present invention further provides pharmaceutically acceptableformulations of the novel compounds of the invention.

The present invention further provides a method for the selectiveelimination of latently infected cells, wherein the cells are latentlyinfected by a lentivirus, especially HIV-1, said treatment comprisingcontacting said cells with an HDACi of the invention in combination withantiretroviral therapy.

What is particularly surprising is that the preferred benzamide HDACi'sof the present invention have been found to be potentiated by buthioninesulfoximine (BSO) at levels of up to 800%. This particularly surprisingas BSO has no appreciable anti-HIV-1 activity, but is capable ofsynergistic action with MS 275, for example, to very substantiallyincrease its ability to kill cells having a latent HIV-1 infection.

This potentiation has the added advantage of reducing the amount ofHDACi and BSO needed for treatment of the patient. For example, both MS275 and BSO have been already tested for safety in humans, and amountsof MS 275 as used for cancer treatment are generally greater than arenecessary for the treatment provided by the present invention. Thepreferred dosage range for MS 275 is in a range of about 0.05-0.1 mg/kgonce weekly, or as prescribed by the physician. BSO may be administeredcontemporaneously with MS 275, at a preferred dosage of 0.1-0.3 mg/kg,and subsequent doses, preferably three to five in number, may beadministered once or twice a day, preferably every 12 h.

Thus, the present invention further provides the use of a histonedeacetylase inhibitor, preferably of the benzamide variety, incombination with buthionine sulfoximine, in the treatment of aretroviral reservoir.

The above-described method for selective elimination of latentlyinfected cells further preferably comprises contacting said cells withBSO in combination with said HDACi, preferably in timings as describedherein.

Although the BSO and HDACi work synergistically, neither is toxic, andthe two may be administered together or separately, provided that bothare present in vivo, particularly preferably in synergistic amounts. BSOmay be formulated with the benzamide, or may be administered separatelyif there are any problems formulating the two active ingredients, or ifthe stability conditions for one are not compatible with the other, forinstance.

As noted above, in general, it has been found that Class I selectiveHDACi show lower toxicity than non-selective compounds, and induceselective killing of HIV-1 infected U1, ACH-2, and H9 IIIB cells,compared with their uninfected counterparts (P<0.01, t-test for slope).

We compared the toxicity of the BSO+HDACi combination in uninfected andlatently infected cell lines. Results showed that, using BSO incombination with HDACis, there was marked cytotoxicity at 72 h ofincubation in latently infected but not in uninfected cell cultures(FIG. 14). Apart from amplifying the effect of histone deacetylaseinhibitors, the results of the present study allow us to hypothesisethat our strategy using pro-oxidant agents such as BSO in combinationwith HDACis is able to induce selective killing of the latently infectedcells.

This strategy can be considered to be one of the long-sought “shock andkill” strategies. These strategies consist of inducing, through drugs,HIV-1 activation from quiescence (i.e. the “shock” phase), in thepresence of ART (to block viral spread), followed by the elimination ofinfected cells (i.e. the “kill” phase), through either natural means(e.g. immune response, viral cytopathogenicity) or artificial means(e.g. drugs, monoclonal antibodies, etc.) [Hamer DH, 2004]. Indeed, ourstrategy is based on an HDACi, which activates HIV-1 replication inlatently infected cells (i.e. the “shock” phase), in combination with apro-oxidant agent such as BSO, which amplifies the HDCAi response andcauses cellular damage due to the HIV-1 induced decay in theintracellular levels of reduced glutatione (i.e. the “kill” phase). Thesearch of a drug combination capable of exerting such effects has, sofar, been a “Holy Grail” in AIDS research.

We have shown herein that gold-containing compounds are useful in thetreatments of the present invention. Surprisingly, we have also foundthat arsenic-containing compounds are similarly useful.

Thus, the invention also provides for the use of an arsenic-containingcompound in the treatment of a retroviral reservoir. Preferably, thecompound is capable of inducing retroviral replication in a recognisedmodel of a reservoir of said retrovirus. The retrovirus may be HIV-1 andthe model may be selected from the U1 and ACH-2 cellular models. Theretrovirus is preferably an HIV virus. Preferably, thearsenic-containing compound is an arsenic oxide, such as As₄O₆, althougharsenic trioxide (As₂O₃) is particularly preferred.

Preferably, the use may further comprise the use of at least one furtheroxidative stress inducer, such as a non-iron metallodrug epigeneiticmodulator, for instance gold-containing compounds, such as auranofin; ahistone deacetylase inhibitor (HDACi); a pro-oxidant molecule, forinstance a glutathione synthesis inhibitor such as BSO; and/or ironnitriloacetate or ferrous sulphate. The HDACi is preferably any of thosedefined above, and particularly from class I HDACi's.

The HDACi's may be:

-   -   a benzamide HDACi having the formula:

in which Y comprises one or two six-membered rings, each beingunsaturated or partially unsaturated and heterocyclic or homocyclic,with two to eight linking atoms, and wherein either the linking atoms ora ring comprises an amino group and a carbonyl group; or

-   -   a hydroxamate HDACi having the formula:

wherein R is a direct bond or an ethylene or ethenylene group, X is═CH—or ═N—, R¹ is aryl or heteroaryl, and is mono- or bi-cyclic, and R²is straight or branched chain alkyl, optionally substituted by a mono-or bi-cyclic aryl or heteroaryl group.

Also provided is a method of treating a patient suspected of having aretroviral reservoir, comprising administering to said patient thearsenic-containing compound, which may also include administration of atleast one further oxidative stress inducer, such as a non-ironmetallodrug epigeneitic modulator, for instance gold-containingcompounds, such as auranofin; a histone deacetylase inhibitor (HDACi); apro-oxidant molecule, for instance a glutathione synthesis inhibitorsuch as BSO; and/or iron nitriloacetate or ferrous sulphate. Patientswho are presenting low CD4 counts are preferred. Said patients may beundergoing, or have undergone, antiretroviral therapy (ART).

All aspects of the present invention may be used in the treatment of thelatent reservoirs of retroviruses, which are present in every retroviralinfection. The present invention may be used in the treatment ofpatients suspected of having latent reservoirs of retroviruses. Suchretroviruses will be referred to herein as HIV-1, although it will beappreciated that this is for convenience, and that all such referencesinclude references to other retroviruses, unless otherwise apparent fromthe context.

The treatment is preferably intended to eliminate any latent reservoirof retrovirus. However, it will be appreciated that such treatments mayalso be used, less preferably, to control latent retrovirus. This may beadvantageous where an individual is particularly susceptible to restartinfections from the reservoirs, for example. The selective killing oflatently infected cells described above is particularly advantageous.

A latent reservoir is a cell or cells containing the retrovirus eitherin quiescence, or replicating at low rates, such as less than 5% of thenormal rate, such that the cell can act to protect the virus for aperiod of time, and often only releases viable virions weeks or monthsafter normal antiretroviral treatment has stopped, thereby requiringcontinued treatment of the individual, when infection recurs throughsuch a reservoir.

The ‘normal’ antiretroviral treatment referred to above, and which isadministered to HIV-1-infected individuals, generally consists of acombination of at least three different drugs belonging to differentclasses, and are normally selected from the nucleosidic/nucleotidicreverse transcriptase inhibitors (NRTI's), the non-nucleosidic reversetranscriptase inhibitors (NNRTI's), and the protease inhibitors (PIs). Afusion inhibitor, or a chemokine receptor blocker may also form a partof the treatment. Recently, a further drug class, the integraseinhibitors, has been added to the arsenal of antiretroviral drugs.Antiretroviral drug combinations, in general, show the capacity to blockongoing viral replication, but have no potential for eradicating thevirus from the body.

Reference herein is made to actives. It will be appreciated that thisrefers to the oxidative stress inducers, as discussed herein.

For the elimination of retroviral reservoirs, the treatment of thepresent invention uses the present actives, together with conventionalanti-retroviral therapy, as well known in the art, and as describedabove, for example.

The actives may be administered direct to the patient in any suitable,pharmaceutically acceptable vehicle, and by any suitable route, as maybe determined by a skilled person or physician. The actives may beadministered orally, or as an injection or by transdermal patch, forexample, and may be in free solution, or bound to a carrier. Otherformulations, such as tablets, suppositories, creams and sprays willgenerally be less useful, although they may be used if deemedappropriate.

Suitable administration may be co-temporaneous or by separate delivery.Suitable vehicles may include targeted liposomes, bearing suitableantibodies, but infected latent cells are largely associated with thecirculation, so administration by intravenous injection is alsoconsidered, although the preferred route of administration for all thecompounds of the present invention is oral.

The present invention further provides a method for the selectiveelimination of latently infected cells, wherein the cells are latentlyinfected by a lentivirus, especially HIV-1, said treatment comprisingcontacting said cells with an HDACi of the invention in combination withantiretroviral therapy.

Potentiation (synergy) has been shown between many of the compounds ofthe present inventions with HDACi's. This has the added advantage ofreducing the amount of actives needed for treatment of the patient.

Furthermore, many of these actives have already been approved for humanadministration or have passed phase I clinical trials for safety.

Thus, the present invention further provides the use of the presentactives in the treatment of a retroviral reservoir and for the selectiveelimination of latently infected cells.

Reference herein to BSO, Auranofin or Arsenic trioxide includes anyanalogues thereof.

It is also envisaged that glutathione synthesis inhibitors such asbuthionine sulfoximine (BSO) can be used alone in the treatment ofretroviral reservoirs, i.e. in treating latently infected cells,preferably by targeting and selectively kill infected, but notun-infected, cells.

Combinations of any of the oxidative stress inducers (non-ironmetallodrug epigeneitic modulators), glutathione synthesis inhibitors orHDACi's described herein can be used.

The HDACi's are most preferably class I HDACi's. Those described in Maiet al 2009 are also preferred and its disclosure is hereby incorporateby reference.

The following Experimental section is for illustration only, and is notlimiting on the present invention.

All references cited herein are herby incorporated by reference to theextent that they do not conflict with the teaching of the presentinvention.

EXAMPLE 1 Auranofin

Methods

HIV-1 induction in ACH-2 and U1 cells. Latently HIV-1 infected ACH-2cells were incubated with the compounds in 96-well plates under standardculture conditions (RPMI medium, 10% foetal bovine serum/FBS, and properantibiotics, which were switched from time to time in order to avoidselection of drug resistant contaminating bacteria), and the content ofHIV-1 p24 core antigen in supernatants was measured at 24 and 72 h ofincubation by HIV-1 p24 ELISA kits (Perkin Elmers, Boston , Mass.),following the Manufacturers instructions. Cell viability was tested foreach treatment after collection of supernatants, as follows.

Cell viability assay. Cell viability was quantitated using the methyltetrazolium (MTT) procedure. Absorbance values were generated at awavelength of 550 nm using 96-well ELISA readers and subtracted of theblank values using cell culture media in the absence of cells. Cellviability in the presence of the test compounds was expressed as apercentage of the cell viability of control untreated cell cultures.

Test for detection of the combined effects of two drugs. For detectionof synergism/antagonism/additivity, cells were incubated with differentconcentrations of either drug alone or both drugs in combination, and,again, viral replication was quantified at three days of incubation byELISA testing.

Flow cytometry. To determine the HIV-1 LTR-controlled expression ofgreen fluorescent protein (GFP) in the Jurkat cell clones, cells werepooled and washed three times in ice-cold PBS with NaN₃ (0.02%) andbovine serum albumin (2%; PBS A/A). Cells were then fixed in 1%paraformaldehyde for 20 min., washed and resuspended in PBS A/A.Fluorescence was then acquired by a flow cytometer (FACScalibur,Becton-Dickinson, Mountain View, Calif.). Fluorescence data werecollected on a 4-decade log scale and the relative fluorescenceintensity was stated as the percentage of fluorescent cells beyond thethreshold value established using non-transfected Jurkat cells.

Detection of NF-kappaB nuclear translocation. Nuclear extracts fromcontrol Jurkat 8.4 cells and cells treated with auranofin at differenttimes of incubation were obtained using a nuclear extraction kit(Chemicon International), following the Manufacturer's instructions.NF-kappaB nuclear translocation was detected using a colorimetrictranscription factor assay (Millipore) for the p65 subunit. Results wereexpressed as a percentage of the signal obtained in cells incubated withthe potent NF-kappaB stimulating cytokine TNF-alpha (5 ng/ml) for 1.5 h,at which the peak of nuclear translocation of NF-kappaB is reached.

Data analysis. Experiments were performed on, at least, two differentoccasions with similar results and results were shown as means. Analyseswere conducted using the GraphPad software. For concentration-dependencecurves, the percentage-of-control values were plotted against thedifferent drug concentrations. An appropriate transformation was appliedto restore normality where necessary. The lines, or curves, best fittingthe data points were generated by the least squares method. Thethreshold for significance was considered to be P=0.05, in case oflinear regression, or R²=0.7, in case of non-linear regression.Non-linear regression was preferred over linear regression (also in casethe latter was significant), where the R² was superior. Differencesbetween drug concentration responses were analyzed using the t-test forslope.

Synergism was analyzed by means of percentage-of-synergism values, whichrepresent the percent difference between the effects of the drugcombination and the sum of the effects of either drug administeredseparately at matched concentrations, calculated as follows:

PS=100·[E _(drug A+drug B)−(E _(drug A) +E _(drug B))]/(E_(drug A) +E_(drug B))

where PS is the percentage of synergism and E is the effect of the drug,expressed as the fold-increase in p24 production.

Three-dimensional (3D) x,y.z graphs were generated by plottingpercentage of synergism values (z axis) against the matched drugconcentrations used (in the x and y axes). 3D surfaces were generatedusing Microsoft Excel. Highly convex surfaces indicate synergism. Flatsurfaces indicate an additive effect, while concave surfaces showantagonism.

Results

HIV-1 activation by auranofin in U1 and ACH-2 cells

To preliminarily assess the HIV-1 activating effects of auranofin incell lines in which the post-integrational stages of HIV-1 replicationare inducible, HIV-1 infected T-lymphoid ACH-2 and monocytic U1 cellswere incubated with increasing concentrations of the compound, and HIV-1replication was measured at 24 h (data not shown) and 72 h of incubation(FIG. 2 shows data from ACH-2 cells). 5 ng/ml of TNF-alpha, a cytokinepotently promoting HIV-1 replication by inducing NF-kappaB (p65/p50)activation was used as a positive control. Results showed that auranofinincreased HIV-1 replication in a time- and dose-dependent manner(P=0.0295, t-test for regression; FIG. 5B) in the 0.125-0.5 μM range ofconcentrations, approximating the mean plasma levels observed duringtreatment of rheumatoid arthritis [Benn et al., 1991].

Of note, the HIV-1 activating effect of auranofin was synergistic tothat of MC2113, a class I HDACI from our institutional library endowedwith poor toxicity [Rotili et al., 2009]. This was shown in experimentsin which auranofin was co-administered with MC 2113 to U1 cells (FIG.3). The effect was visible as soon as at 24 h of incubation.

The Cellular Basis for the Auranofin Response

To evaluate the auranofin response within a cellular population, we usedthe latently infected T-lymphoid Jurkat cell clone 8.4, established byJordan et al. [Jordan et al., 2003]. This cell clone contains the entireHIV-1 genome under control of the LTR and presenting the GFP genereplacing nef. As opposed to U1 cells, these cells displaynon-significant basal levels of HIV-1 expression and have a functionalTat/TAR axis. In the 8.4 cells, auranofin induced a dose-dependent shiftin fluorescence (FIG. 4), which was evident mostly at the highestconcentrations adopted. The cellular basis for the additive effects ofthe HDACI/auranofin combination was also explored. We found thataddition of auranofin recruited the HDACI-unresponsive cells into theresponding cell population (FIG. 5).

Similar results were obtained in other cell lines expressing GFP undercontrol of HIV-1 LTR and using other HDACi such as suberoylanilidehydroxamic acid (SAHA). Jurkat A1 cells have a green fluorescent protein(GFP) gene under control of HIV-1 LTR, which is quiescent in a portionof the cell population. These cells were treated with a clinicallyrelevant concentration of auranofin (0.25 μM) or 1 μM of MC2113, orboth. Data was presented as the percentage of fluorescent cells beyond athreshold established using non-GFP expressing Jurkat cells. This datademonstrated induction of LTR-controlled expression of green-fluorescentprotein (GFP) by auranofin and a non-class-specific histone deacetylaseinhibitor (SAHA).

Auranofin-Induced NF-kappaB Nuclear Translocation

It is well known that oxidative stress causes activation and nuclearlocalization of the NF-kappaB heterodimer Rel A (p65)/p50 [Rahman etal., 2004], which is important for HIV-1 transcription and replication[Williams et al., 2007]. If auranofin should activate HIV-1 fromquiescence by inducing an oxidative stress within target cells, then,NF-kappa B (p65/p50) nuclear translocation should be visible. To testthis hypothesis, aliquots of nuclear extracts from Jurkat 8.4 cellstreated with auranofin (250 μM) were subjected to a colorimetric assayfor the p65 (RelA) subunit of NF-kappaB. Results showed a time-dependentNF-kappaB accumulation within the nuclei (FIG. 6). We conclude thatauranofin induces NF-kappaB (p65/p50) activation under conditionssimilar to those at which it activates HIV-1 from quiescence.

Synergistic Effects of Auranofin with Other Pro-Oxidant Strategies

To gain some insight into the auranofin-induced HIV-1 activation fromquiescence, the effects of the drug were tested in the presence of somewell-characterized pro-oxidant molecules, i.e., iron nitriloacetate(FeNTA) [Savarino et al., 1999], which promotes oxidative stress throughthe Fenton reaction, and buthionine sulfoximine (BSO), an irreversibleinhibitor of gamma-glutamyl cysteine synthetase, a limiting enzyme inthe glutathione synthetic pathway [Anderson, 1998]. Results showed that,similarly to auranofin, FeNTA dose-dependently induced HIV-1 replicationin ACH-2 cells (data not shown), whereas BSO alone had no effect onHIV-1 inducing effects at concentrations up to 500 μM (data not shown).When co-administered with either of the two agents, auranofin exertedsynergistic HIV-1 activating effects, as shown by the highly convexsurfaces in the percentage-of-synergism graphs (FIGS. 7 and 8). Weconclude that auranofin potentiates iron-induced HIV-1 activation andthat the effects of this drug are enhanced by glutathione depletion.

References (all references cited herein are hereby incorporated to theextent that they do not conflict withy the present invention).

Anderson M E: Glutathione: an overview of biosynthesis and modulation.Chem Biol Interact 1998, 111-112:1-14.

Benn H P, Schnier C, Bauer E, Seiler K U, Elhöft H, Löffler H. Biliary,renal and fecal elimination and distribution of gold after a single oraladministration of auranofin, quantified by the instrumental neutronactivation analysis method. Z Rheumatol. 1991 Jan-Feb;50(1):32-8.

Chouchane S, Snow E T. In vitro effect of arsenical compounds onglutathione-related enzymes. Chem Res Toxicol. 2001 May;14(5):517-22.

Daniel L W, Civoli F, Rogers M A, Smitherman P K, Raju P A, Roederer M.ET-18-OCH3 inhibits nuclear factor-kappa B activation by12-O-tetradecanoylphorbol-13-acetate but not by tumor necrosisfactor-alpha or interleukin 1 alpha. Cancer Res. 1995 Nov. 1;55(21):4844-9.22. Chircorian A, Barrios A M, Inhibition of lysosomalcysteine proteases by chrysotherapeutic compounds: a possible mechanismfor the antiarthritic activity of Au(I), Bioorg. Med. Chem. Lett. 14(2004), pp. 5113-5116.

Demonté D, Quivy V, Colette Y, Van Lint C: Administration of HDACinhibitors to reactivate HIV-1 expression in latent cellular reservoirs:implications for the development of therapeutic strategies. BiochemPharmacol 2004, 68:1231-1238.

Dueñas-González A, Garcia-López P, Herrera L A, Medina-Franco J L,González-Fierro A, Candelaria M. The prince and the pauper. A tale ofanticancer targeted agents. Mol Cancer. 2008 Oct. 23; 7:82.

Duverger A, Jones J, May J, Bibollet-Ruche F, Wagner F A, Cron R Q,Kutsch O: Determinants of the establishment of human immunodeficiencyvirus type 1 latency. J Virol 2009, 83:3078-93.

Hamer D H: Can HIV be Cured? Mechanisms of HIV persistence andstrategies to combat it. Curr HIV Res. 2004, 2:99-111.

Huang R, Wallqvist A, Covell D G. Anticancer metal compounds in NCI'stumor-screening database: putative mode of action. Biochem Pharmacol.2005 Apr. 1; 69(7):1009-39.

Israël N, Gougerot-Pocidalo M A: Oxidative stress in humanimmunodeficiency virus infection. Cell Mol Life Sci 1997, 53:864-70.

Jeon K I, Jeong J Y, Jue D M. Thiol-reactive metal compounds inhibitNF-kappa B activation by blocking I kappa B kinase. J Immunol. 2000 Jun1;164(11):5981-9.

Jordan A, Bisgrove D, Verdin E: HIV reproducibly establishes a latentinfection after acute infection of T cells in vitro. EMBO J 2003,22:1868-77.

Kalantari P, Narayan V, Natarajan S K, Muralidhar K, Gandhi U H, VuntaH, Henderson A J, Prabhu K S. Thioredoxin reductase-1 negativelyregulates HIV-1 transactivating protein Tat-dependent transcription inhuman macrophages. J Biol Chem. 2008 Nov. 28; 283(48):33183-90.

Lu J, Chew E H, Holmgren A. Targeting thioredoxin reductase is a basisfor cancer therapy by arsenic trioxide. Proc Natl Acad Sci U S A. 2007

Lu J, Holmgren A. Selenoproteins. J Biol Chem. 2009 Jan. 9;284(2):723-7.

Omata Y, Folan M, Shaw M, Messer R L, Lockwood P E, Hobbs D, BouillaguetS, Sano H, Lewis J B, Wataha J C.Sublethal concentrations of diversegold compounds inhibit mammalian cytosolic thioredoxin reductase(TrxR1).Toxicol In Vitro. 2006 Sep;20(6):882-90.

Patai S. The Chemistry of Organic Derivatives of Gold and Silver. Editedby Saul Patai and Zvi Rappoport 1999 John Wiley & Sons, Ltd., passim.

Rahman I, Marwick J, Kirkham P: Redox modulation of chromatinremodeling: impact on histone acetylation and deacetylation, NF-kappaBand pro-inflammatory gene expression. Biochem Pharmacol 2004,68:1255-67.

Rigobello M P, Scutari G, Boscolo R, Bindoli A. Induction ofmitochondrial permeability transition by auranofin, a gold(I)-phosphinederivative. Br J Pharmacol. 2002 August; 136(8):1162-8.

Rigobello M P, Scutari G, Folda A, Bindoli A. Mitochondrial thioredoxinreductase inhibition by gold(I) compounds and concurrent stimulation ofpermeability transition and release of cytochrome c. Biochem Pharmacol.2004 Feb. 15; 67(4):689-96.

Rotili D, Simonetti G, Savarino A, Palamara A T, Migliaccio A R, Mai A:Non-cancer uses of histone deacetylase inhibitors: effects on infectiousdiseases and β-hemoglobinopathies. Curr Topics Med Chem 2009,9(3):272-91.

Sannella A R, Casini A, Gabbiani C, Messori L, Bilia A R, Vincieri F F,Majori G, Severini C. New uses for old drugs. Auranofin, a clinicallyestablished antiarthritic metallodrug, exhibits potent antimalarialeffects in vitro: Mechanistic and pharmacological implications. FEBSLett. 2008 Mar. 19; 582(6):844-7.

Savarino A, Bottarel F, Malavasi F, Dianzani U. Role of CD38 in HIV-1infection: an epiphenomenon of T-cell activation or an active player invirus/host interactions? AIDS. 2000 Jun. 16; 14(9):1079-89.

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EXAMPLE 2 HDACi's

HIV-1 infected lymphocytic ACH-2 and monocytoid U1 cells (bothdisplaying low baseline levels of HIV-1 replication) are wellestablished cell-line models for HIV-1 latency [Munier et al., 2005].They were incubated with the test compounds in 96-well plates understandard culture conditions, and HIV-1 replication was measured at 72 hof incubation by ELISA testing. The potency of the compounds on HIV-1induction was assessed as the fold-increase in HIV-1 replication in thepresence of a standard concentration of 1 μM of the test compound (thisconcentration is generally used as a threshold for lead compoundsselection), as compared to the baseline levels observed in untreatedcontrols. We found several compounds capable of inducing HIV-1replication in latently infected cells, and, in general, there was goodagreement between results in ACH-2 and U1 cells.

Several compounds (Class I selective or non-selective HDACi) showedremarkable HIV-1 stimulatory activity (Table 1), which correlated withinfected cell killing (U1 cells: P=0.0378; ACH-2 cells: P=0.0017;Spearman's non-parametric test (FIG. 9 A,B)). Class I selectivecompounds, in general, showed lower toxicity than non-selectivecompounds (data not shown). Moreover, some Class I-selective HDACipromoting HIV-1 reactivation induced selective killing of HIV-1-infectedcells. This is exemplified by data using MC1855 (P<0.01; t-test forslope; FIG. 9 C,D).

From the structure/activity relationships, particular requirementsemerge for efficient induction of HIV-1 escape from latency. In general,HDACi display a general pharmacophoric model, which comprises a capgroup (CAP) able to interact with the rim of the catalytic tunnel, oftenwith a polar connection unit (CU), linking the CAP to a hydrophobicspacer (HS), which allows the molecule to lie into the tunnel [Mai etal., 2005b]. Finally, the HS carries at the end a Zn²⁺ binding group(ZBG) that is able to complex the Zn²⁺ at the bottom of the cavity [Maiet al., 2005b]. Generally the ZBG consists of a hydroxamate,carboxylate, or a benzamide.

MS 275 is a Class-I selective HDACi in clinical trials for cancertreatment [Nishioka et al., 2008; Hauschild et al., 2008]. This compoundproved the most potent inducer of HIV-1 escape from latency among thecompounds tested, displaying activity in the nanomolar range in ACH-2(FIGS. 10) and U1 cells (not shown), well within the plasmaconcentrations achievable in vivo [Zhao et al., 2007]. Our SAR studiesare not intended to limit the chemical types of HADACi administrablewith butionine sulfoximine (BSO), and synergistic effects (see below) ofHDACi with BSO are extendable to the entire HDACi class.

BSO which induces oxidative stress, was also tested. BSO, an inhibitorof gamma glutamyl cysteine synthetase [Garaci et al., 1996] (a limitingenzyme in glutathione synthesis), promotes oxidative stress indirectlyby decreasing the cellular antioxidant defence [Anderson, 1998]. Thiscompound was tested because it might favour NF-kappa B activation, whichis enhanced in an oxidative environment. Results showed that BSO did notsignificantly induce HIV-1 replication in either ACH-2 or U1 cells (datanot shown). Despite this, BSO was tested to determine whether it mightpotentiate the effects of HDACi. Results showed that BSO strikinglyincreased the HIV-1 promoting effects of two benzamide HDACi, i.e. MS275 and MC2211, as shown in FIGS. 11 and 12. The highly convex 3Dsurfaces of the percentage-of-synergism graph in ACH-2 cells point to asynergistic effect of the drug combinations. The BSO concentrationsshowing the synergistic effect are achievable therapeutically [Lacretaet al., 1994]. Similar results were obtained in U1 cells (data notshown).

The finding that Class I HDAC inhibition is per se sufficient for HIV-1reactivation is particularly advantageous.

Methods

Chemistry. Melting points were determined on a Buchi 530 melting pointapparatus and are uncorrected. Infrared (IR) spectra (KBr) were recordedon a Perkin-Elmer Spectrum One instrument. ¹H NMR spectra were recordedat 400 MHz on a Bruker AC 400 spectrometer; chemical shifts are reportedin E(ppm) units relative to the internal reference, tetramethylsilane(Me₄Si). All compounds were routinely checked by TLC and ¹H NMR. TLC wasperformed on aluminium-backed silica gel plates (Merck D C, AlufolienKieselgel 60 F₂₅₄) with spots visualised by UV light. All solvents werereagent grade and, when necessary, were purified and dried by standardmethods. The concentration of solutions after reactions and extractionsinvolved the use of a rotary evaporator operating at reduced pressure ofca. 20 Torr. Organic solutions were dried over anhydrous sodiumsulphate. Analytical results are within ±0.40% of the theoreticalvalues. A SAHA sample for biological assays was prepared as wellaccording to standard methods. All chemicals were purchased from AldrichChimica, Milan (Italy) or from Lancaster Synthesis GmbH, Milan (Italy)and were of the highest purity [Mai et al. 2006].

General Procedure for the Synthesis of Ethyl Esters of4-(3,4-Dihydro-4-oxo-6-substituted-2-pyrimidinylthio)methylcinnamicAcids. Example: Ethyl Ester of4-(3,4-Dihydro-4-oxo-6-benzyl-2-pyrimidinylthio)methylcinnamic Acid. Amixture of 6-benzyl-4-hydroxy-2-mercaptopyrimidine (6.87 mmol, 1.5 g),crude ethyl 4-bromomethylcinnamate (7.56 mmol, 2.2 g), and anhydrouspotassium carbonate (7.56 mmol, 1.0 g) in 3 mL of anhydrous DMF wasstirred at room temperature for 1 h. After treatment with cold water(100 mL), the aqueous phase was extracted with ethyl acetate (3×40 mL).The organic phase was washed with brine (3×40 mL), dried, and evaporatedto dryness to furnish the crude desired compound, which was purified bychromatography on a silica gel column, eluting with a mixture ethylacetate/hexane (1:1) to give the desired product as a white solid (1.2g). ¹H NMR (CDCl₃) δ1.33 (t, 3H, CH₂CH₃), 3.83 (s, 2H, PhCH₂), 4.26 (q,2H, CH₂CH₃), 4.38 (s, 2H, CH₂S), 5.98 (s, 1H, C₅-H), 6.40 (d, 1H,CH═CHCO), 7.33 (m, 9H, two benzene rings), 7.61 (d, 1H, CH═CHCO), 13.20(s, 1H, NH). Anal. C, H, N, S [Mai et al. 2006].

General Procedure for the Synthesis of6-(3,4-Dihydro-4-oxo-6-(un)substituted-2-pyrimidinylthio)-hexanoic Acidsand 4-(3,4-Dihydro-6-substituted-4-oxopyrimidin-2-ylthio)methylcinnamicAcids. Example: 6-(3,4-Dihydro-4-oxopyrimidin-2-ylthio)hexanoic Acid. Amixture of the appropriate ethyl ester(1.1 mmol, 0.3 g), 2 N KOH (8.8mmol, 0.49 g), and EtOH (5 mL) was stirred at room temperature for 18 h.The solution was poured into water (50 mL) and extracted with ethylacetate (2×20 mL). HCI (2 N) was added to the aqueous layer until the pH5, and the precipitate was filtered and recrystallised to yield thetitle compound (0.23 g) as a pure solid. ¹H NMR (DMSO-d₆) δ1.32 (m, 2H,CH₂CH₂CH₂S), 1.49 (m, 2H, CH₂CH₂CO), 1.61 (m, 2H, CH₂CH₂S), 1.93 (t, 2H,CH₂CO), 3.06 (t, 2H, CH₂S), 6.07 (s, 1H, C₅-H), 7.83 (s, 1H, C₆-H), 12.2(s, 1H, COOH). Anal. C, H, N, S [Mai et al. 2006].

General Procedure for the Synthesis ofN-Hydroxy-6-(3,4-dihydro-4-oxo-6-(un)substituted-2-pyrimidinylthio)hexanamidesandN-Hydroxy-4-(3,4-dihydro-6-substituted-4-oxopyrimidin-2-ylthio)-methylcinnamylamides.Example: N-Hydroxy-6-(3,4-dihydro-4-oxopyrimidin-2-ylthio)hexanamide. Toa 0° C. cooled solution of the appropriate carboxylic acid (0.9 mmol,0.22 g) in dry tetrahydrofuran (5 mL), ethyl chloroformate (2.2 mmol,0.21 mL) and triethylamine (2.3 mmol, 0.33 mL) were added, and themixture was stirred for 10 min. The solid was filtered off, and to thefiltrate was added O-(2-methoxy-2-propyl)hydroxylamine (5.4 mmol, 0.4mL). The resulting mixture was stirred at room temperature for 1 h, thenit was evaporated under reduced pressure, and the residue was diluted inMeOH (5 mL). Amberlyst 15 ion-exchange resin (0.18 g) was added to thesolution of the O-protected hydroxamate, and the mixture was stirred atroom temperature for 1 h. Afterward, the reaction was filtered, and thefiltrate was concentrated in a vacuum to give crude final product, whichwas purified by crystallization. ¹H NMR (DMSO-d₆) δ1.30 (m, 2H,CH₂CH₂CH₂S), 1.46 (m, 2H, CH₂CH₂CO), 1.60 (m, 2H, CH₂CH₂S), 1.90 (t, 2H,CH₂CO), 3.02 (t, 2H, CH₂S), 6.10 (s, 1H, C₅-H), 7.85 (s, 1H, C₆-H), 8.66(s, 1H, NHOH), 10.33 (s, 1H, NHOH), 12.5 (s, 1H, uracil NH). Anal. C, H,N, S [Mai et al. 2006].

General procedure for the synthesis of3-(4-acylaminophenyI)-N-hydroxy-2-propenamides. Example:344-(2,3-Diphenylpropionylamino)phenyn-N-hydroxy-2-propenamide (MC1895).

Ethyl chloroformate (1.26 mmol, 0.12 mL) and triethylamine (1.37 mmol,0.19 mL) were added to a cooled (0° C.) solution of3-[4-(2,3-diphenylpropionylamino)phenyl]-2-propenoic acid (1.05 mmol,0.39 g) in dry THF (10 mL), and the mixture was stirred for 10 min. Thesolid was filtered off, and O-(2-methoxy-2-propyl)hydroxylamine (3.15mmol, 0.23 mL) was added to the filtrate. The solution was stirred for15 min at 0° C., then was evaporated under reduced pressure, and theresidue was diluted in methanol (10 mL). Amberlyst® 15 ion-exchangeresin (105 mg) was added to the solution of the 0-protected hydroxamate,and the mixture was stirred at room temperature for 1 h. After, thereaction was filtered and the filtrate was concentrated in vacuo to givethe crude MC1895 which was purified by crystallization. ¹H NMR (DMSO-d₆)δ3.05 (m, 1H, PhCH₂), δ3.60 (m, 1H, PhCH₂), δ3.75 (m, 1H, PhCHCO), δ6.36(d, 1H, PhCH═CHCOOEt), δ7.15-7.70 (m, 15H, benzene protons andPhCH═CHCOOEt), δ9.00 (s, 1H, OH), δ10.23 (s, 1H, CONHPh), δ10.85 (s, 1H,NHOH). Anal. C, H, N, O.

Maize HD2, HD1-B, and HD1-A Enzyme Inhibition in Vitro. Radioactivelylabelled chicken core histones were used as the enzyme substrateaccording to established procedures [referenced in: Mai et al., 2006].The enzyme liberated tritiated acetic acid from the substrate, which wasquantified by scintillation counting. The IC₅₀ values are the results oftriple determinations. A 50 μL sample of maize enzyme (at 30° C.) wasincubated (30 min) with 10 microL of total [³H]acetate-prelabelledchicken reticulocyte histones (2 mg/mL). The reaction was stopped by theaddition of 36 microL of 1 M HCl/0.4 M acetate and 800 microL of ethylacetate. After centrifugation (10 000 g, 5 min), an aliquot of 600microL of the upper phase was counted for radioactivity in 3 mL ofliquid scintillation cocktail. The compounds were tested at a startingconcentration of 40 microM, and active substances were diluted further.TSA and SAHA were used as the reference compounds, and blank solventswere used as negative controls [Mai et al. 2006].

Mouse HDAC1 Enzyme Assay. For the inhibition assay, partially purifiedHDAC1 from mouse liver (anion exchange chromatography) was used as theenzyme source. HDAC activity was determined using[³H]acetate-prelabelled chicken reticulocyte histones as the substrate.Mouse HDAC1 (50 microL) was incubated with different concentrations ofcompounds for 15 min on ice, and 10 μL of total [³H]acetate-prelabelledchicken reticulocyte histones (2 mg/mL) were added, resulting in aconcentration of 41 microM. The mixture was incubated at 37° C. for 1 h.The reaction was stopped by the addition of 50 microL of 1 M HCl/0.4 Macetylacetate and 1 mL ethyl acetate. After centrifugation at 10 000 gfor 5 min, an aliquot of 600 microL of the upper phase was counted forradioactivity in a 3 mL liquid scintillation cocktail [Mai et al. 2006].

Cellular Assays. Cell Lines and Cultures. The U937 cell line wascultured in RPMI with 10% foetal calf serum, 100 U/mL of penicillin, 100microg/mL of streptomycin, and 250 ng/mL of amphotericin-B, 10 mM HEPESand 2 mM glutamine. U937 cells were kept at the constant concentrationof 200 000 cells per millilitre of culture medium. Human breast cancerZR-75.1 cells were propagated in DMEM medium supplemented with 10%foetal calf serum and antibiotics (100 U/mL of penicillin, 100micrograms/mL of streptomycin, and 250 ng/mL of amphotericin-B).

Ligands and Materials. SAHA was dissolved in DMSO and used at 1 or 5microM. MS-275 (gift from Schering AG) was dissolved in ethanol and usedat 5 microM. UBHA compounds 1d and 1j were dissolved in DMSO and used at1 or 5 microM.

Cell-Based Human HDAC1 and HDAC4 Assays. Cells (U937 cells for the HDAC1assay and ZR75.1 cells for the HDAC4 assay) were lysed in IP buffer (50mM Tris-HCl at pH 7.0, 180 mM NaCl, 0.15% NP-40, 10% glycerol, 1.5 mMMgCl₂, 1 mM NaMO₄, and 0.5 mM NaF) with a protease inhibitor cocktail(Sigma), 1 mM DTT, and 0.2 mM PMSF for 10 min in ice and centrifuged at13 000 rpm for 30 min. Then, 1000 micrograms of extracts were diluted inIP buffer up to 1 mL and pre-cleared by incubating with 20 microL of A/Gplus Agarose (Santa Cruz) for 30 min to 1 h on a rocking table at 4° C.Supernatants were transferred into a new tube, and the antibodies(around 3 to 4 pg) were added and IP was allowed to proceed overnight at4° C. on a rocking table. The antibodies used were HDAC1 (Abcam) andHDAC4 (Sigma). As the negative control, the same amount of proteinextracts were immunoprecipitated with the corresponding purified IgG(Santa Cruz). On the next day, 20 microL of A/G and Agarose (Santa Cruz)were added to each IP, and incubation was continued for 2 h. The beadswere recovered by brief centrifugation and washed with cold IP bufferseveral times. The samples were than washed twice in PBS andre-suspended in 20 microL of sterile PBS. The HDAC assay was carried outaccording to the suppliers instructions (Upstate). Briefly, samplesimmunoprecipitated with the HDAC4 and HDAC1 or with purified IgG werepooled separately to homogenise all samples. Then, 10 microL of the IPwas incubated with a previously labelled ³H-Histone H4 peptide linkedwith streptavidine agarose beads (Upstate). In detail, 120 000 CPM ofthe H4-³H-acetyl-peptide was used for each tube and incubated in 1× HDACbuffer with 10 microL of the sample in the presence or absence of HDACinhibitors with a final volume of 200 μL. Those samples were incubatedovernight at 37° C. in slow rotation. On the next day, 50 μL of aquenching solution was added, and 100 microL of the samples were countedin duplicate after brief centrifugation in a scintillation counter.Experiments have been carried out in quadruplicate [Mai et al., 2006].

Compound preservation. Compounds were preserved as dry powders, andresuspended in dimethyl sulphoxide (DMSO) at the moment of use. Initialconcentrations in DMSO solutions were adjusted in order to obtain properdrug concentrations with less than 2/1000 DMSO (v/v) in the final cellculture media.

HIV-1 reactivation screening test. Latently HIV-1 infected ACH-2 cellswere incubated with the compounds in 96-well plates under standardculture conditions (RPMI medium, 10% foetal bovine serum/FBS, and properantibiotics, changed from time to time in order to avoid selection ofdrug resistant contaminating bacteria), and the content of HIV-1 p24core antigen in supernatants was measured at 72 h of incubation by HIV-1p24 ELISA kits (from Perkin Elmer), following the Manufacturersinstructions. The potency of the compounds on induction of HIV-1 escapefrom latency was assessed as the fold-increase (%) in HIV-1 replicationin the presence of a standard concentration of 1 microM, as compared tothe baseline levels observed in untreated controls. The 1 μMconcentration was chosen because it is generally considered to be athreshold for selection of lead compounds. Cell viability was tested foreach treatment after collection of supernatants, as follows.

Cell viability assay. Cell viability was quantitated using the highlystandardised methyl tetrazolium (MTT) procedure (described in detail in:Savarino A, Calosso L, Piragino A, Martini C, Gennero L, Pescarmona G P,Pugliese A. Modulation of surface transferrin receptors in lymphoidcells de novo infected with human immunodeficiency virus type-1.CellBiochem Funct. 1999 March; 17(1):47-55). Absorbance values weregenerated at a wavelength of 450 nm using 96-well ELISA readers andsubtracted of the blank values using cell culture media in the absenceof cells. Cell viability in the presence of the test compounds wasexpressed as a percentage of the cell viability of control untreatedcell cultures. For testing of selective killing, Non-infected H9, andU937, and HIV-1 infected H9_(IIIB), ACH-2, and U1 cells were incubatedwith the test compounds for seven days, and cell viability was measuredas described above.

Concentration-response curves. Concentration-response curves weregenerated for those compounds with the best activities, by incubatingcells with decreasing concentrations of the compounds (0.001-1 μM).

Test for detection of combined drug effects. For detection ofsynergy/antagonism, cells were incubated with different combinations ofBSO alone, the histone deacetylase inhibitor alone, or both, and, again,viral replication was quantified at three days of incubation by ELISAtesting.

Data analysis. Experiments were performed on at least two differentoccasions with similar results and results were shown as means. Analyseswere conducted using the GraphPad software. Correlation between celldeath/HIV-1 reactivation was assessed by plotting, for each compound,the percentage of inhibition of cell viability against the fold-increasein HIV-1 replication. Significance of the correlation was assessed usingSpearman's correlation coefficients using a significance threshold ofP=0.05. For concentration-dependence curves, the fold-increase valueswere plotted against the different concentrations. An appropriatetransformation was applied to restore normality where necessary. Thelines, or curves, best fitting the data points were generated by theleast squares method. The threshold for significance was considered tobe P=0.05, in case of linear regression, or R²=0.7, in case ofnon-linear regression. Synergism was analysed by means ofpercentage-of-synergism values, which represent the percent differencebetween the effects of the drug combination and the sum of the effectsof the histone deacetylase inhibitor and the pro-oxidant drugadministered separately at matched concentrations, calculated asfollows:

PS=100·[E _(drug A+drug B)−(E _(drug A) +E _(drug B)i)]/(E _(drug A) +E_(drug B))

where PS is the percentage of synergism and E is the effect of the drugconcentration, expressed as the fold-increase in p24 production.

Three-dimensional (3D) x,y.z graphs were generated by plottingpercentage of synergism values (z axis) against the matched drugconcentrations used (in the x and y axes). 3D surfaces were generatedusing Microsoft Excel. Highly convex surfaces indicate synergism. Flatsurfaces indicate an additive effect, while concave surfaces showantagonism.

TABLE 1 Structures and potencies of different compounds in inducingHIV-1 replication in lymphocytic ACH-2 and monocytoid U1 cells. Foldincrease (%) in HIV-1 p24 at 1 μM (unless otherwise specified) Com-HDACs Chemical ACH-2 U1 pound Structure inhibited class cells cells MC1855

Class I Hydroxamates 699 817 MC 2111

Class I Hydroxamates 2,460 2,960 MC 2113

Class I Hydroxamates 3,304 2,846 MC 2195

Class I Hydroxamates 3,427 4,076 MC 1895

Class I Hydroxamates 2,981 3,747 MC 1857

NS Hydroxamates 1,655. 880 MC 1864

NS Hydroxamates 1,145 995 MS 275

Class I Benzamides 23,162 1,835 MC 2211

Class I Benzamides 2,025 4,811

TABLE 2 HDAC inhibiting activity of cited MC compounds. % inhibitionIC₅₀ values, nM @ 5 μM Maize Maize mouse IP IP Compound Ref. HD1-B HD1-AHDAC1 HDAC1 HDAC4 MC1855 162 55 128 78.7 0 MC2111 58 42 620 73.6 0MC2113 62 69 560 64.9 22.4 MC2195 11 8 69 46.7 93.1 MC2211* Mai et al.,2008 MC1857 72 80 291 79.3 N.D. MC1864 89 64 305 55.0 60.4 MC1895 133 7483 72.5 16.1 MS 65.4 0 275** N.D., Not determined *tested on humanrecombinant HDAC1 and 4. **confirmed by literature data using tested onhuman recombinant HDACs.

References for HDACi's:

-   Anderson M E. Glutathione: an overview of biosynthesis and    modulation. Chem Biol Interact. 1998 Apr. 24; 111-112:1-14.-   Annemieke J. M. de Ruijter, Albert H. Van Gennip, Huib N. Caron et    all. Histone deacetylases (HDACs): Characterization of the classical    HDAC family. Biochem. J. 2003. 370, 737-749.-   Benn H P, Schnier C, Bauer E, Seiler K U, Elhöft H, Loftier    H.[Biliary, renal and fecal elimination and distribution of gold    after a single oral administration of auranofin, quantified by the    instrumental neutron activation analysis method]Z Rheumatol. 1991    January-February; 50(1):32-8.-   Biochem J. 2008 Jan. 15; 409(2):581-9.-   Bowie A, O'Neill L A. Oxidative stress and nuclear factor-kappaB    activation: a reassessment of the evidence in the light of recent    discoveries. Biochem Pharmacol. 2000 Jan. 1; 59(1):13-23.-   Demonté D, Quivy V, Colette Y, Van Lint C. Administration of HDAC    inhibitors to reactivate HIV-1 expression in latent cellular    reservoirs: implications for the development of therapeutic    strategies. Biochem Pharmacol. 2004 Sep. 15; 68(6):1231-8.-   Dokmanovic M, Clarke C, Marks P A. Histone deacetylase inhibitors:    overview and perspectives. Mol Cancer Res. 2007 October;    5(10):981-9.-   Duong, V.; Bret, C.; Altucci, L.; Mai, A.; Duraffourd, C.;    Loubersac, J.; Harmand, P.; Bonnet, S.; Valente, S.; Maudelonde, T.;    Caveillés, V.; Boulle, N. Specific regulation and activity of class    II histone deacetylases in human breast cancer cells. Mol. Cancer    Res. 2008, in press.-   Garaci E, Palamara A T, Ciriolo M R, D'Agostini C, Abdel-Latif MS,    Aquaro S, Lafavia E, Rotilio G. Intracellular GSH content and HIV    replication in human macrophages. J Leukoc Biol. 1997 July;    62(1):54-9.-   Hamer D H. Can HIV be Cured? Mechanisms of HIV persistence and    strategies to combat it. Curr HIV Res. 2004 April; 2(2):99-111.-   Hauschild A, Trefzer U, Garbe C, Kaehler K C, Ugurel S, Kiecker F,    Eigentler T, Krissel H, Schott A, Schadendorf D. Multicenter phase    II trial of the histone deacetylase inhibitor    pyridylmethyl-N-{4-[(2-aminophenyl)-carbamoyq-benzyl}-carbamate in    pretreated metastatic melanoma. Melanoma Res. 2008 August;    18(4):274-8.-   Hezareh M. Prostratin as a new therapeutic agent targeting HIV viral    reservoirs. Drug News Perspect. 2005 October; 18(8):496-500.-   Hulgan T, Morrow J, D'Aquila RT, Raffanti S, Morgan M, Rebeiro P,    Haas D W. Oxidant stress is increased during treatment of human    immunodeficiency virus infection. Clin Infect Dis. 2003 Dec. 15;    37(12):1711-7.-   Illi, B.; Dello Russo, C.; Colussi, C.; Rosati, J.; Pallaoro, M.;    Spallotta, F.; Rotili, D.; Valente, S.; Ragone, G.; Martelli, F.;    Biglioli, P.; Steinkuhler, C.; Gallinari, P.; Mai, A.;    Capogrossi, M. C.; Gaetano, C. Nitric oxide modulates chromatin    folding in human endothelial cells via protein phosphatase 2A    activation and class II histone deacetylases nuclear shuttling. Circ    Res. 2008, 102, 51-58.-   Inoue, S.; Mai, A.; Dyer, M. J. S.; Cohen, G. M. Inhibition of    histone deacetylase class I but not class II is critical for the    sensitization of leukemic cells to tumor necrosis factor-related    apoptosis-inducing ligand-induced apoptosis. Cancer Res. 2006, 66,    6785-6792.-   Jiang G, Espeseth A, Hazuda D J, Margolis D M. c-Myc and Sp1    contribute to proviral latency by recruiting histone deacetylase 1    to the human immunodeficiency virus type 1 promoter. J Virol. 2007    October; 81(20):10914-23.-   Khan N, Jeffers M, Kumar S, Hackett C, Boldog F, Khramtsov N, Qian    X, Mills E, Berghs S C, Carey N, Finn P W, Collins L S, Tumber A,    Ritchie J W, Jensen P B, Lichenstein H S, Sehested M.-   Lacreta F P, Brennan J M, Hamilton T C, Ozols RF, O'Dwyer P J.    Stereoselective pharmacokinetics of L-buthionine SR-sulfoximine in    patients with cancer. Drug Metab Dispos. 1994 November-December;    22(6):835-42.-   Lehrman G, Hogue I B, Palmer S, Jennings C, Spina C A, Wiegand A,    Landay A L, Coombs R W, Richman D D, Mellors J W, Coffin J M, Bosch    R J, Margolis D M. Depletion of latent HIV-1 infection in vivo: a    proof-of-concept study. Lancet. 2005 Aug. 13-19; 366(9485):523-4.-   a Mai A, Massa S, Ragno R, Cerbara 1, Jesacher F, Loidl P, Brosch G.    3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-alkylamides as a new    class of synthetic histone deacetylase inhibitors. 1. Design,    synthesis, biological evaluation, and binding mode studies performed    through three different docking procedures. J Med Chem. 2003 Feb.    13; 46(4):512-24.-   b Mai, A.; Massa, S.; Pezzi, R.; Rotili, D.; Loidl, P.; Brosch, G.    Discovery of (aryloxopropenyl)pyrrolyl hydroxyamides as selective    inhibitors of class Ila histone deacetylase homologue HD1-A. J. Med.    Chem. 2003, 46, 4826-4829.-   a Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso,    A.; Scognamiglio, A.; Altucci, L.; Loidl, P.; Brosch, G. Class 11    (IIa)-selective histone deacetylase inhibitors. 1. Synthesis and    biological evaluation of novel (aryloxopropenyl)pyrrolyl    hydroxyamides. J. Med. Chem. 2005, 48, 3344-3353.-   b Mai, A.; Massa, S.; Rotili, D.; Pezzi, R.; Bottoni, P.; Scatena,    R.; Meraner, J.; Brosch, G. Exploring the connection unit in the    HDAC inhibitor pharmacophore model: novel uracil-based hydroxamates.    Bioorg. Med. Chem. Lett. 2005, 15, 4656-4661.-   Mai A, Massa S, Rotili D, Simeoni S, Ragno R, Botta G, Nebbioso A,    Miceli M, Altucci L, Brosch G. Synthesis and biological properties    of novel, uracil-containing histone deacetylase inhibitors. J Med    Chem. 2006 Oct. 5; 49(20):6046-56.-   Mai A, Perrone A, Nebbioso A, Rotili D, Valente S, Tardugno M, Massa    S, De Bellis F, Altucci L. Novel uracil-based 2-aminoanilide and    2-aminoanilide-like derivatives: histone deacetylase inhibition and    in-cell activities. Bioorg Med Chem Lett. 2008 Apr. 15;    18(8):2530-5.-   Mottet D, Castronovo V. Histone deacetylases: a new class of    efficient anti-tumor drugs. Med Sci (Paris). 2008 August-September;    24(8-9):742-6.-   Munier S, Delcroix-Genête D, Carthagéna L, Gumez A, Hazan    U.Characterization of two candidate genes, NCoA3 and IRF8,    potentially involved in the control of HIV-1 latency.Retrovirology.    2005 Nov. 23; 2:73.-   Nishioka C, Ikezoe T, Yang J, Takeuchi S, Koeffler HP, Yokoyama A. M    S-275, a novel histone deacetylase inhibitor with selectivity    against HDAC1, induces degradation of FLT3 via inhibition of    chaperone function of heat shock protein 90 in AML cells.Leuk Res.    2008 September; 32(9):1382-92.-   Palamara A T, Garaci E, Rotilio G, Ciriolo M R, Casabianca A,    Fraternale A, Rossi L, Schiavano G F, Chiarantini L, Magnani M.    Inhibition of murine AIDS by reduced glutathione. AIDS Res Hum    Retroviruses. 1996 Sep. 20; 12(14):1373-81.-   Ragno R, Simeoni S, Rotili D, Caroli A, Botta G, Brosch G, Massa S,    Mai A. Class II-selective histone deacetylase inhibitors. Part 2:    alignment-independent GRIND 3-D QSAR, homology and docking studies.    Eur J Med Chem. 2008 March; 43(3):621-32.-   Saari H, Suomalainen K, Lindy O, Konttinen Y T, Sorsa T. Activation    of latent human neutrophil collagenase by reactive oxygen species    and serine proteases. Biochem Biophys Res Commun. 1990 Sep. 28;    171(3):979-87.-   Sannella A R, Casini A, Gabbiani C, Messori L, Bilia A R, Vincieri F    F, Majori G, Severini C. New uses for old drugs. Auranofin, a    clinically established antiarthritic metallodrug, exhibits potent    antimalarial effects in vitro: Mechanistic and pharmacological    implications. FEBS Lett. 2008 Mar. 19; 582(6):844-7.-   Savarino A, Pescarmona G P, Boelaert J R. Iron metabolism and HIV    infection: reciprocal interactions with potentially harmful    consequences? Cell Biochem Funct. 1999 December; 17(4):279-87.-   Savarino A, Pistello M, D'Ostilio D, Zabogli E, Taglia F, Mancini F,    Ferro S, Matteucci D, De Luca L, Barreca M L, Ciervo A, Chimirri A,    Ciccozzi M, Bendinelli M. Human immunodeficiency virus integrase    inhibitors efficiently suppress feline immunodeficiency virus    replication in vitro and provide a rationale to redesign    antiretroviral treatment for feline AIDS. Retrovirology. 2007 Oct.    30; 4:79.-   Scognamiglio, A., Nebbioso, A., Manzo, F., Valente, S., Mai, A.,    Altucci, L. HDAC-class II specific inhibition involves HDAC    proteasome-dependent degradation mediated by RANBP2. Biochim Biophys    Acta 2008 released on web 2008 Jul. 22.-   Smith S M. Valproic acid and HIV-1 latency: beyond the sound bite.    Retrovirology. 2005 Sep. 19; 2:56.-   Williams S A, Chen L F, Kwon H, Ruiz-Jarabo C M, Verdin E, Greene    W C. NF-kappaB p50 promotes HIV latency through HDAC recruitment and    repression of transcriptional initiation. EMBO J. 2006 Jan. 11;    25(1):139-49.-   Zhao M, Rudek M A, Mnasakanyan A, Hartke C, Pili R, Baker S D. A    liquid chromatography/tandem mass spectrometry assay to quantitate    MS-275 in human plasma. J Pharm Biomed Anal. 2007 Jan. 17;    43(2):784-7.

EXAMPLE 3 Further Work on HDACI's+BSO

To investigate the cellular basis of the synergism between HDACi andBSO, we used the Jurkat model for HIV-1 quiescence. These results arederived from the A1 Jurkat cell clone, which has an integrated GFP/Tatconstruct under control of the HIV-1 LTR, which is quiescent in themajority of cells and thus allows us to examine, by flow cytometry,activation of the LTR promoter at a single-cell level [Jordan A, et al2003]. Our results showed that BSO recruited HDACI-insensitive cellsinto the responding cell population (FIG. 13). Differently from theresults obtained from p24 measurements in ACH-2 and U1 cells, BSO aloneinduced LTR activation in a small proportion of cells.

HIV-1 replicating cell cultures display decreased levels of reducedglutathione [Simon G, et al 1994]. We compared the toxicity of theBSO+HDACi combination in uninfected and latently infected cell lines.Results showed that, using BSO in combination with HDACis, there wasmarked cytotoxicity at 72 h of incubation in latently infected but notin uninfected cell cultures (FIG. 14).

This is supported by experiments in uninfected Jurkat cells and Jurkatcell clones (6.3 and 8.4), which contain a quiescent HIV-1 genome (withthe GFP gene) under control of the LTR [Jordan A, 2003, infra]. We foundthat the 6.3 cell clone succumbed more readily to the MS-275/BSOcombination than its uninfected counterpart (FIG. 14). Similar resultswere obtained with the 8.4 clone (data not shown).

In conclusion, the effects of BSO allow us to amplify the proportion ofcells responding to an HDACi-based HIV-1 reactivating treatment. Ingeneral, oxidative stress tilts the balance of HAT/HDAC activity towardsincreased HAT activity and DNA unwinding, thus facilitating the bindingof several transcription factors [Rahman I, et al 2004]. This suggestsimportant clinical uses, because a variegated phenotype afteractivation, with only a fraction of the cell population becomingactivated in response to a global signal, was also shown by Jordan etal. 2004 [infra], who attributed this phenomenon to the different localchromatin environments.

Apart from amplifying the effect of histone deacetylase inhibitors, theresults of the present study allow us to hypothesise that our strategyusing pro-oxidant agents such as BSO in combination with HDACis is ableto induce selective killing of the latently infected cells. Thisstrategy can be considered to be one of the long-sought “shock and kill”strategies. These strategies consist of inducing, through drugs, HIV-1activation from quiescence (i.e. the “shock” phase), in the presence ofART (to block viral spread), followed by the elimination of infectedcells (i.e. the “kill” phase), through either natural means (e.g. immuneresponse, viral cytopathogenicity) or artificial means (e.g. drugs,monoclonal antibodies, etc.) [Hamer DH, 2004]. Indeed, our strategy isbased on an HDACi, which activates HIV-1 replication in latentlyinfected cells (i.e. the “shock” phase), in combination with apro-oxidant agent such as BSO, which amplifies the cellular damage dueto the HIV-1 induced decay in the intracellular levels of reducedglutatione (i.e. the “kill” phase). The search of a drug combinationcapable of exerting such effects has, so far, been a “Holy Grail” inAIDS research.

References for Example 3

-   Jordan A, Bisgrove D, Verdin E: HIV reproducibly establishes a    latent infection after acute infection of T cells in vitro. EMBO J    2003, 22:1868-1877.-   Simon G, Moog C, Obert G: Valproic acid reduces the intracellular    level of glutathione and stimulates human immunodeficiency virus.    Chem Biol Interact 1994, 91:111-121.-   Rahman I, Marwick J, Kirkham P: Redox modulation of chromatin    remodeling: impact on histone acetylation and deacetylation,    NF-kappaB and pro-inflammatory gene expression. Biochem Pharmacol    2004, 68:1255-1267.-   Hamer D H: Can HIV be Cured? Mechanisms of HIV persistence and    strategies to combat it. Curr HIV Res 2004, 2:99-111.

EXAMPLE 4 Arsenic Trioxide

Gold salts and arsenicals share a number of biological effects, and arenow showing new promise as potentially promising epigenetic metallodrugswith several applications in medicine. Their therapeutic use in otherdisease conditions has an ancient tradition, of course. Although arsenicis considered to be an element displaying both metal and non-metalproperties, it behaves as a metal in many aspects. In its most commonelemental form (gray arsenic), it has a metal-like nature and conductselectricity. Similar to gold salts, arsenicals have been employedtherapeutically since the 5^(th) century BC. Hippocrates used realgar(As₂S₂) and orpiment (As₂S₃) as remedies for ulcers. Traditional Chinesemedicine used arsenicals, in combination with gold to treat variousconditions. Olympiodorus of Thebes (5th century AD) roasted arsenicsulfide and obtained white arsenic (As₂O₃, but may also be found asAs₄O₆). Both gold salts and arsenicals later found, among other uses,antirheumatic, antisyphilitic and antitumour applications [see Eisler,2003, Giordano, 1844, Christison and Griffith, 1848, Cutler andBradford, 1878, Waxman et al., 2001, Park et al., 2008]. Arsenic is alsoknown as a poison, when used at higher dosage.

The use of arsenic trioxide and gold salts was gradually abandoned inthe past century, limiting the use of arsenic trioxide to chronicpromyelocytic leukemia and gold salts to rheumatoid arthritis. In thelast decade, however, interest for these types of drugs has beenrefuelled in light of the discovery of their epigenetic effects, i.e.the capacity to induce modifications of the DNA structure withoutaltering the sequence of bases. Structural modifications of DNA such aswinding/unwinding regulate gene expression. Further research showed thatarsenic trioxide and gold salts share important similarities in theireffects at the cellular level, which might account for the similartherapeutic applications that both types of drugs have found duringtheir history. Both arsenic trioxide and gold salts induce reactiveoxygen species (ROS).

Oxidative stress is known to tilt the balance of HAT/HDAC activitytowards increased HAT activity and DNA unwinding, thus facilitating thebinding of several transcription factors [Rahman et al., 2004].Interestingly, both arsenic trioxide and gold-containing compoundsinhibit thioredoxin reductase and act as superoxide dismutase (SOD)mimics. Therefore, in light of these effects, and common epigeneticeffects such as induction of HAT activity, gold compounds and arsenicalsare starting to be considered as a unique drug class [Talbot et al.,2008].

This drug class, comprising arsenicals and gold-containing compounds,will be henceforth dubbed “epigenetic metallodrugs” i.e. metallodrugshaving epigenetic effects. Other metallodrugs known to activate HIV-1from latency, such as iron-containing compounds and cisplatin [Savarinoet al., 1998; Spandidos et al., 1990], do not show thepro-differentiating effects of gold compounds and arsenicals.

Moreover, gold and arsenic ions share the ability to block the activesite of thioredoxin reductases (TrxR) by complexing directly with theselenocystein residue important for the reducing activity of theseproteins. This activity of auranofin is shared with arsenic trioxide[Liu et al. 2007]. The gold(I) derivatives and arsenic trioxide appearas very good inhibitors of thioredoxin reductase, exhibiting IC₅₀ valuesin the nanomolar range (IC₅₀<300 nM), whereas metal ions and metalcomplexes are active in the micromolar range (from 19 to 80 μM)[Bragadin et al., 2004; Lu et al., 2007]. Another activity shared byarsenic trioxide and a gold(I) derivative such as auranofin is theunique ability to suppress synthesis of TrxRs [Talbot et al., 2008].

We have shown in Example 1 that the gold-containing compound, auranofinis able to induce HIV-1 escape from latency in vitro at concentrationsmimicking those found in plasma of humans with rheumatoid arthritis. Ifour theory is correct, the effects of the gold-containing compoundauranofin on HIV-1 escape from latency should also be exerted byarsenicals.

Arsenic trioxide has well documented epigenetic potential, a welldescribed toxicity profile and its clinical use is relatively safe atcertain dosages.

We first tested the response to arsenic trioxide in Jurkat cell cloneswith an integrated green fluorescence protein (GFP)-encoding gene undercontrol of the HIV-1 LTR [Jordan et al., 2003].

The experiments were conducted according to the techniques extensivelydescribed Example 1. In these Jurkat cell clones, GFP induction byHDACIs was evident only in a fraction of cells and increased in responseto arsenic trioxide in a concentration-dependent manner (FIG. 15).

To evaluate the response to arsenic trioxide within a cellularpopulation containing the entire HIV-1 genome, we used the latentlyinfected T-lymphoid Jurkat cell clones 6.3 and 8.4, established byJordan et al. [Jordan et al., 2003]. This cell clones contain the entireHIV-1 genome under control of the LTR and presenting the GFP genereplacing nef. As opposed to U1 cells, these cells displaynon-significant basal levels of HIV-1 expression and have a functionalTat/TAR axis. In the 8.4 cells, arsenic trioxide induced adose-dependent shift in fluorescence (data not shown), which was evidentmostly at the highest concentrations adopted.

We conclude that the effect of arsenic trioxide replicate those ofauranofin in cell line models for HIV-1 latency. Similar to auranofin,cisplatin was capable of activating latent HIV-1 at concentrations inthe nanomolar range. Auranofin and arsenic trioxide do not share theireffects with other metallodrugs such as cisplatin, which activatesquiescent HIV-1 in the micromolar rather than in the nanomolar range ofconcentrations [Spandidos et al., 1990]. Our results thus support theview that gold(I) derivatives and arsenic trioxide should be regarded asbelonging to a unique drug class of epigenetic modulators. Similar toauranofin, arsenic trioxide might find an application in “smoking out”strategies to purge HIV-1 from reservoirs.

We have shown in Examples 2 and 3 that a “shock and kill” effect can beobtained in cell line models for HIV-1 latency by combining Class Ihistone deacetylase inhibitors (HDACIs) with buthionine sulfoximine, aninhibitor of gamma-glutamyl cysteine synthetase, i.e. a limiting enzymefor glutathione synthesis [Savarino et al., 2009].

Glutatione is undoubtedly one important antioxidant defense, however,BSO alone was incapable to induce HIV-1 escape from latency only poorly.This is likely to be explained by the fact that BSO does not per secause an oxidative stress but, rather, impairs the ability of a cellpopulation to counteract the oxidative stress induced by HDACIs[Palamara et al., unpublished data]. In this context, auranofin andarsenic trioxide are per se capable of inducing oxidative stress likelythrough their superoxide-dismutase mimicking effects and capable ofcounteracting the antioxidant defense by inhibiting TrxRs. These effectscould represent a step forward in the exploitation of oxidative stressas a means to combat HIV-1 latency.

Recently, Chomont et al. identified transitional memory T-CD4⁺cells(T_(TM)s) as the principal reservoir for HIV-1 latency in individualsunder antiretroviral therapy (ART) and presenting low CD4 counts[Chomont et al., 2009]. T_(TM)s are precursors of central memory T CD4⁺cells (T_(CM)s), which represent a more stable HIV-1 reservoir andsurvive for years. As compared to Toms, T_(TM)s present a lessdifferentiated phenotype and proliferate in response to IL-7, a cytokineinducing stem cell proliferation. In order to obtain HIV-1 eradicationfrom ART-treated individuals with low CD4 counts, Chomont et al.advocate treatment with an “intelligent-targeted chemotherapy”, which,in combination with ART, be able to inhibit T-cell proliferation anddecrease stem cell-ness in order to avoid generation of the long lastingT_(CM) reservoir. The authors however were unable to identify suitabledrug candidates. However, the HIV-1 inducing effects of arsenic trioxide(allowing elimination of the infected cells by viral cytopathogenicityor the immune system), added to its well-known antiblastic effects onlymphocytes as well as its marked pro-differentiating activity wouldmake this drug an ideal candidate for clinical trials of HIV-1eradication.

References for Example 4

-   Bragadin M, Scutari G, Folda A, Bindoli A, Rigobello M P. Effect of    metal complexes on thioredoxin reductase and the regulation of    mitochondrial permeability conditions. Ann N Y Acad Sci. 2004    Dec;1030:348-54.-   Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio F A,    Yassine-Diab B, Boucher G, Boulassel M R, Ghattas G, Brenchley J M,    Schacker T W, Hill B J, Douek D C, Routy J P, Haddad E K, Sekaly    R P. HIV reservoir size and persistence are driven by T cell    survival and homeostatic proliferation. Nat Med. 2009 August;    15(8):893-900.-   Cutler E G, Bradford E H. Action of iron, cod-lived oil and arsenic    on the globular richness of the blood. Am J Med Sci 1878; 75:74-84.-   Christison and Griffith's Dispensatory 1848 (A Dispensatory or    Commentary on the Pharmacopeias of Great Britan and the United    States). Lea and Blanchard publishers of Philadelphia.-   Eisler R. Chrysotherapy: a synoptic review. Inflamm Res. 2003    December; 52(12):487-501.-   Giordano A. Farmacologia, ossia trattato di farmacia teorico e    pratico. Torino, 1844 Ed. Zecchi e Bona, Contrada Carlo Alberto.-   Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a    latent infection after acute infection of T cells in vitro. EMBO J.    2003;22:1868-1877.-   Lu J, Chew E H, Holmgren A. Targeting thioredoxin reductase is a    basis for cancer therapy by arsenic trioxide. Proc Natl Acad Sci U    S A. 2007 Jul. 24; 104(30):12288-93.-   Park S J, Kim M, Kim N H, Oh MK, Cho J K, Jin J Y, Kim I S.    Auranofin promotes retinoic acid- or dihydroxyvitamin D3-mediated    cell differentiation of promyelocytic leukaemia cells by increasing    histone acetylation. Br J Pharmacol. 2008 July; 154(6):1196-205.-   Rahman I, Marwick J, Kirkham P: Redox modulation of chromatin    remodelling: impact on histone acetylation and deacetylation,    NF-kappaB and pro-inflammatory gene expression. Biochem Pharmacol    2004, 68:1255-1267-   Savarino A, Mai A, Norelli S, E I Daker S, Valente S, Rotili D,    Altucci L, Palamara A T, Garaci E. “Shock and kill” effects of class    I-selective histone deacetylase inhibitors in combination with the    glutathione synthesis inhibitor buthionine sulfoximine in cell line    models for HIV-1 quiescence. Retrovirology. 2009 Jun. 2; 6:52.-   Spandidos D A, Zoumpourlis V, Kotsinas A, Maurer H R,    Patsilinacos P. Transcriptional activation of the human    immunodeficiency virus long terminal repeat sequences by cis-platin.    Genet Anal Tech Appl. 1990 September; 7(5):138-41.-   Talbot S, Nelson R, Self WT. Arsenic trioxide and auranofin inhibit    selenoprotein synthesis: implications for chemotherapy for acute    promyelocytic leukaemia.Br J Pharmacol. 2008 July; 154(5):940-8.-   Waxman S, Anderson K C. History of the development of arsenic    derivatives in cancer therapy. The Oncologist, Vol. 6, Suppl 2,    3-10, April 2001.

1.-20. (canceled)
 21. A method for selectively targeting a cell latentlyinfected with human immunodeficiency virus (HIV), which method comprisescontacting a cell with arsenic trioxide in an amount effective forinducing replication of the latent HIV, wherein the arsenic trioxide hasan epigenetic effect on the latently infected cell.
 22. A methodaccording to claim 21, in which the arsenic trioxide is administered incombination with at least one compound selected from the groupconsisting of: a histone deacetylase inhibitor (HDACI), butioninesulfoximine (BSO), iron nitriloacetate (FeNTA), and ferrous sulphate.23. A method according to claim 21, in which the cell is selected fromthe group consisting of an ACH-2 cell and a U1 cell.
 24. A methodaccording to claim 21, additionally comprising administration of aconventional anti-retroviral therapy.
 25. A method for treating aretroviral infection in an individual, which method comprisesadministering, to the individual, arsenic trioxide in an amounteffective for inducing replication of latent HIV, wherein the arsenictrioxide has an epigenetic effect.
 26. A method according to claim 25,wherein the arsenic trioxide is administered in combination with atleast one compound selected from the group consisting of: a histonedeacetylase inhibitor (HDCAI), butionine sulfoximine (BSO), ironnitriloacetate (FeNTA) and ferrous sulphate.
 27. A method according toclaim 25 additionally comprising administration of a conventionalanti-retroviral therapy.
 28. A method according to claim 25, wherein thearsenic trioxide is administered in combination with suberoylanilidehydroxamic acid (SAHA).
 29. A method according to claim 22, wherein thehistone deacetylase inhibitor (HDACI) is suberoylanilide hydroxamic acid(SAHA).
 30. A method according to claim 26, wherein the histonedeacetylase inhibitor (HDACI) is suberoylanilide hydroxamic acid (SAHA).